High purity x-chromosome bearing and y-chromosome bearing populations of spermatozoa

ABSTRACT

Isolated non-naturally occurring populations of spermatozoa ( 15 ) having high purity and technologies to differentiate spermatozoa ( 28 ) based on characteristics such as mass, volume, orientation, or emitted light including methods of analysis and apparatus such as beam shaping optics ( 30 ) and detectors ( 32 ).

I. TECHNICAL FIELD

[0001] Isolated high purity X-chromosome bearing or Y-chromosome bearingpopulations of spermatozoa and technologies to isolate spermatozoa,particles, or events based upon differentiation characteristics such asmass, volume, DNA content, or the like.

II. BACKGROUND

[0002] Isolated high purity X-chromosome bearing or Y-chromosome bearingpopulations of spermatozoa can be utilized to accomplish in vitro or invivo artificial insemination of or fertilization of ova or oocytes ofnumerous mammals such as bovids, equids, ovids, goats, swine, dogs,cats, camels, elephants, oxen, buffalo, or the like. See also, U.S. Pat.No. 5,135,759, hereby incorporated by reference.

[0003] However, conventional technologies for separating spermatozoainto X-chromosome bearing and Y-chromosome bearing populations canresult in spermatozoa populations having low purity. Regardless of theseparation method spermatozoa have not been routinely separated intoX-chromosome bearing and to Y-chromosome bearing sperm samples havinghigh purity, such as 90%, 95%, or greater than 95%.

[0004] A number of techniques, directly or indirectly based ondifferences in size, mass, or density have been disclosed with respectto separating X-chromosome bearing from Y-chromosome bearing spermatozoaAs disclosed by U.S. Pat. No. 4,474,875, a buoyant force is applied toall sperm cells simultaneously and X-chromosome bearing and Y-chromosomebearing spermatozoa may then be isolated at different locations in theseparation medium. U.S. Pat. No. 5,514,537 discloses a technique wherebyspermatozoa traverse a column packed with two sizes of beads. The largerX-chromosome bearing spermatozoa become isolated in the layer containingthe larger beads, while the smaller Y-chromosome bearing spermatozoabecome isolated in the layer containing the smaller beads. U.S. Pat. No.4,605,558 discloses that spermatozoa may be made differentiallyresponsive to a density gradient and U.S. Pat. No. 4,009,260 exploitsthe differences in migration-rate, or swimming-speed, between theY-bearing spermatozoa, and the X-chromosome bearing spermatozoa, througha column of retarding medium.

[0005] A problem common to each of the above-mentioned technologies maybe that they each act on all the spermatozoa in a ‘bulk-manner’, meaningthat all the spermatozoa undergo the same treatment at the same time,and the Y-chromosome bearing sperm cells come out faster, earlier, or ata different position than X-chromosome bearing sperm cells. As such,individual sperm cells may not be assessed and there may be no actual‘measurement’ of volume, mass, density, or other sperm cellcharacteristics. One-by-one assessment of sperm cells can provideadvantages in that the actual separation process can be monitored, andobjective quantitative data can be generated even during the separationprocess, and separation parameters altered as desired. Furthermore,these technologies may not be coupled with flow cell sorting devices.

[0006] Flow cytometer techniques for the separation of spermatozoa havealso been disclosed. Using these techniques spermatozoa may be stainedwith a fluorochrome and made to flow in a narrow stream or band passingby an excitation or irradiation source such as a laser beam. As stainedparticles or cells pass through the excitation or irradiation source,the fluorochrome emits fluorescent light. The fluorescent light may becollected by an optical lens assembly, focused on a detector, such as aphotomultiplier tube which generates and multiplies an electronicsignal, which may then be analyzed by an analyzer. The data can then bedisplayed as multiple or single parameter chromatograms or histograms.The number of cells and fluorescence per cell may be used ascoordinates. See U.S. Pat. No. 5,135,759, hereby incorporated byreference. However, with respect to this type of technology a variety ofproblems remain unresolved and isolating highly purified populations ofX-chromosome bearing or Y-chromosome bearing sperm cells be difficult.

[0007] A significant problem with conventional flow cytometertechnologies can be the orientation of objects, particles, or cells inthe sheath fluid stream. This can be particularly problematic when theobject or cell is irregular in shape with respect to more than one axis,such spermatozoa for example. One aspect of this problem may beestablishing the initial orientation of the object within the sheathfluid stream. A second aspect of this problem may be maintaining theorientation of the object with respect to the detector (photomultipliertube or otherwise) during the period that emitted light from the objectis measured.

[0008] Another significant problem with conventional flow cytometertechnologies can be the failure to encapsulate the objects or cells in adroplet of liquid. Especially, when droplets are formed aroundirregularly shaped objects the droplet may not be of sufficient size tocompletely surround all the features of the objects or cells. Forexample, during flow cytometry operation as above-described droplets canbe formed at very high speed, even as many as 10,000 to 90,000 dropletsper second and in some applications as many as 80,000 droplets persecond. When spermatozoa are encapsulated into droplets, especially atthese high rates of speed, a portion of the tail or neck may not beencapsulated in the droplet. That portion of the tail or neck notencapsulated in the droplet may then be responsive with the nozzle ormay be responsive to the environment surrounding the droplet in a mannerthat interferes with subsequent droplet formation or with properdeflection of the droplet. As a result some of the spermatozoa may notbe analyzed at all reducing the efficiency of the procedure, or may notbe resolved sufficiently to be assigned to a population, or may bedeflected in errant trajectories, or a combination of all may occur.

[0009] Another significant problem with conventional flow cytometertechnologies, as well as other technologies, can be a coincidence ofmeasurable events. One aspect of this problem can be that the incidentlight flux from a first event continues to produce signals after theincident light flux from a second event starts to generate a signal. Assuch, the two events remain at least partially unresolved from oneanother. Another aspect of this problem can be that two or more eventsare simultaneously initiated and the incident light flux comprises thecontribution of all the events. As such, the multiplicity of events maynot be resolved at all and the objects corresponding to the multiplicityof events can be incorrectly assigned to a population or not assigned toa population at all, or both. Specifically, with respect to flowcytometry, individual particles, objects, cells, or spermatozoa insuspension flow through a beam of light with which they interactproviding a measurable response, such as fluorescent emission. Inconventional flow cytometry, Hoechst stained spermatozoa traverse alaser beam resulting in a fluorescent light emission. The fluorescentlight emission from the excited fluorochrome bound to the DNA can bebright enough to produce an electron flow in conventionalphotomultiplier tubes for a period of time after the actual emissionevent has ended. Moreover, in a conventional flow cytometer, the laserbeam can produce a pattern having a height of 30 μm while the width canbe approximately 80 μm. The nucleus of a bovine spermatozoa whichcontains fluorochrome bound DNA can be about 9 μm in length making theheight of the laser beam some three (3) times greater than the nucleus.This difference can allow for the laser excitation of the boundfluorochrome in more than one spermatozoa within the laser beam patternat one time. Each of these conventional flow cytometry problemsdecreases the ability to resolve individual events from one another.

[0010] Another significant problem with conventional flow cytometertechnologies, and other technologies, can be that irregularly shapedobjects, such as spermatozoa, generate differing signals (shape,duration, or amount) depending on their orientation within theexcitation/detection path. As such, individuals within a homogenouspopulation can generate a broad spectrum of emission characteristicsthat may overlap with the emission characteristics of individuals fromanother homogenous population obviating or reducing the ability toresolve the individuals of the two populations.

[0011] Another significant problem with conventional flow cytometertechnologies, and other technologies, can be that objects are notuniformly exposed to the excitation source. Conventional beam shapingoptics may not provide uniform exposure to laser light when the objectsare close to the periphery of the beam.

[0012] Another significant problem with conventional flow cytometertechnologies can be that objects, such as spermatozoa, can be exposed tothe excitation source for unnecessarily long periods of time.Irradiation of cells, such as spermatozoa, with laser light may resultin damage to the cells or to the DNA contained within them.

[0013] Another significant problem with conventional flow cytometertechnologies can be that there may be a disruption of the laminar flowwithin the nozzle by the injection tube. Disruption of the laminar flowcan change the orientation of irregularly shaped objects within the flowand lower the speed of sorting and the purity of the sorted populationsof X-chromosome bearing sperm or Y-chromosome bearing spermatozoa.

[0014] There may be additional problems with technologies that utilizestain bound to the nuclear DNA of sperm cells. First, because the DNA inthe nucleus is highly condensed and flat in shape, stoichiometricstaining of the DNA may be difficult or impossible. Second, stainednuclei may have a high index of refraction. Third, stain bound to theDNA to form a DNA-stain complex may reduce fertilization rates or theviability of the subsequent embryos. Fourth, the DNA-stain complex istypically irradiated with ultra-violet light to cause the stain tofluoresce. This irradiation may affect the viability of the spermatozoa.Due to these various problems, it may be preferable to use a method thatrequires less or no stain, or less or no ultra-violet radiation, or lessor none of both.

[0015] With respect to generating high purity samples of X-chromosomebearing sperm cell or Y-chromosome bearing sperm cell populations(whether live, fixed, viable, non-viable, intact, tailless, or asnuclei), or generally, with respect to detecting small differences inphoto-generated signal between serial events having relatively highincident light flux, or with respect to orienting irregularly shapedobjects in a fluid stream, or eliminating coincident events within anoptical path, or removing undesirably oriented objects from analysis,the instant invention addresses every one of the above-mentionedproblems in a practical fashion.

III. DISCLOSURE OF THE INVENTION

[0016] A broad object of the invention can be to provide isolated highpurity X-chromosome bearing and Y-chromosome bearing populations ofspermatozoa. Isolated non-naturally occurring populations of spermatozoathat have high purity have numerous applications including sex selectionof offspring from mammals, various in vitro protocols for thefertilization of ova, various in vivo protocols such as artificialinsemination, business methods involving the production of prize animalsor meat animals, or preservation of rare or endangered animals, torecite but a few of the applications for high purity populations ofspermatozoa.

[0017] Another broad object of the invention involves both devices andmethods for the production of high purity X-chromosome bearing andY-chromosome bearing sperm samples.

[0018] Particular embodiments of the invention are described, which maybe used in numerous applications as above-mentioned, that can be used toachieve the specific objects of differentiating between brightphotoemissive events having small measurable differences in total lightflux, orienting irregularly shaped objects in a fluid stream, theminimization of coincident events within an optical path, the removal ofsignal contributed by undesired unoriented objects within an opticalpath (including the removal of the object itself), and the encapsulationof irregularly shaped objects within a droplet. As such, the specificobjects of the invention can be quite varied.

[0019] Another broad object of the invention can be to provideX-chromosome bearing or Y-chromosome bearing spermatozoa samples (live,fixed, viable, non-viable, intact, tailless, or sperm nuclei) having agraded level of high purity in the range of 80%, 85%, 90%, 95%, or evengreater than 95%.

[0020] Another significant object of particular embodiments of theinvention can be to sort spermatozoa into X-chromosome bearing andY-chromosome bearing populations having high purity even at highseparation rates. The high speed separation can produce live sperm ofeach sex at rates of about 500, 1000, 2000, 3000, 4000, 5000, 6000,7000, 8,000, 9,000 or even 10,000 per second, or higher.

[0021] Another significant object of particular embodiments of theinvention can be to substantially eliminate or remove spermatozoa (live,fixed, viable, non-viable, intact, tailless, or sperm nuclei) havingundesired orientation in the excitation/detection portion of the flowpath of a flow cytometer.

[0022] Another significant object of particular embodiments of theinvention can be to provide artificial insemination samples ofX-chromosome bearing or Y-chromosome bearing spermatozoa having a highlevel of purity.

[0023] Another significant object of particular embodiments of theinvention can be to provide in vitro insemination samples ofX-chromosome bearing or Y-chromosome bearing spermatozoa having a highlevel of purity.

[0024] Another significant object of a particular embodiment of theinvention can be to preselect the sex of offspring of femalesinseminated with high purity artificial insemination samples, the sex ofoffspring of ova fertilized with high purity artificial inseminationsamples, with selection success rates of 80%, 85%, 90%, 95%, or greaterthan 95%.

[0025] Another significant object of particular embodiments of theinvention can be to differentiate between photoemissive events havingsmall differences in total emitted light flux.

[0026] Another significant object of particular embodiments of theinvention can be to substantially eliminate or reduce the amount ofbackground noise generated by a photomultiplier tube, even in theabsence of light, during the period after exposure to high incidentlight flux.

[0027] Another significant object of particular embodiments of theinvention can be to substantially eliminate saturation of thephotocathode of photomultiplier tube(s) used in conjunction with flowcytometry, or otherwise.

[0028] Another significant object of particular embodiments of theinvention can be to reduce the number electrons migrating from thephotocathode of a photomultiplier tube to the first dynode.

[0029] Another significant object of particular embodiments of theinvention can be to reduce the total flow of electrons to the Nelectrode of a photomultiplier tube.

[0030] Another significant object of particular embodiments of theinvention can be to allow increased light flux to the photocathode ofthe photomultiplier tube without proportionately increasing the amountof background signal generated by the photomultiplier tube.

[0031] Another significant object of particular embodiments of theinvention can be to increase the signal to background signal ratio frommeasured photoemissive events.

[0032] Another significant object of particular embodiments of theinvention can be to allow increased amplification of the signalgenerated from the photomultiplier tube during high incident light fluxevents or serial high incident light flux events without saturating thephotocathode of the photomultiplier tube.

[0033] Another significant object of particular embodiments of theinvention can be to increase the apparent resolution of chromatograms orhistograms resulting from sorting fluorochrome stained sperm, or othercells, or other objects, having small differences in emitted light fluxupon excitation of the bound fluorochrome(s).

[0034] Another significant object of particular embodiments of theinvention can be to improve the calibration of sorting flow cytometerinstruments when used for sorting spermatozoa.

[0035] Another significant object of particular embodiments of theinvention can be to increase the sperm sorting rate of flow cytometersystems.

[0036] Another significant object of particular embodiments of theinvention can be to increase the purity of the sperm samples sorted byflow cytometry.

[0037] Another significant object of particular embodiments of theinvention can be to provide techniques for the sorting of X-chromosomebearing sperm from Y-chromosome bearing sperm where there is a smalldifference in the amount of Y chromosome DNA to the amount of Xchromosome DNA relative to the total amount of nuclear DNA.

[0038] Another significant object of particular embodiments of theinvention can be to provide techniques which improve the apparentresolution of histograms generated during the process of sortingX-chromosome bearing sperm from Y-chromosome bearing sperm with a flowcytometer.

[0039] Another significant object of particular embodiments of theinvention can be to provide beam shaping optics which minimizescoincidence of objects within the excitation/detection path.

[0040] Another significant object of particular embodiments of theinvention can be to provide beam shaping optics that minimizes the totallumens an object is exposed to traversing the excitation beam. Oneaspect of this object can be to decrease the total lumens an object isexposed to. A second aspect of this object can be to increase the powerof the light source without increasing the total lumens the object isexposed to.

[0041] Another significant object of particular embodiments of theinvention can be to provide beam shaping optics that allow for uniformexposure of objects that pass through the optical path.

[0042] Another significant object of particular embodiments of theinvention can be to provide a nozzle that orients irregularly shapedobjects in a fluid stream. One aspect this object can be to orientelongated objects in the same direction. A second aspect of this objectcan be to orient dorso-laterally flatted objects in the same direction.

[0043] Another significant object of particular embodiments of theinvention can be to fully encapsulate irregularly shaped objects withina drop of fluid.

[0044] Another significant object of particular embodiments of theinvention can be to differentiate undesirably oriented objects fromdesirably oriented objects in a fluid stream.

[0045] Another object of an embodiment of the invention can be toprovide differential interference contrast technology, whereby theobject-plane consists of a fluid stream carrying the objects ofinterest, and whereby the image-plane can be used to measure the signalfrom the passing objects.

[0046] Another object of an embodiment of the invention can be toprovide optics that form two laterally separated images from each objectin such a way that one can be used to measure the actual volume, and oneto determine the orientation. This way, objects that were not orientatedproperly to allow a accurate measurement of its volume can be discarded.This can be accomplished by modifications so that the light pulses,resulting from these two images can be detected independently using twopinholes in the image plane. Optics are tuned in such a way that a firstimage can give rise to a light pulse proportional to the volume of theobject, and that a second image can give rise to a light pulse dependenton the orientation the object had when it was measured.

[0047] Another object of an embodiment of the invention can provide amanner of compensating for the fact that the objects are containedinside a fluid stream. The fluid stream can be a cylinder of water, forexample, which acts as a cylindrical lens, thus distorting the image ofthe object. Optically, this corresponds to cylinder of higher refractiveindex (water) than its surroundings (air). The compensation disclosed inthis invention can consist of, for example, a cylinder having arefractive index lower than its surroundings, although othercompensating elements of various shapes and refractive index may also bedesigned as the need requires. By making sure the light passes throughthis compensation element, the optical effect of the fluid stream can becompensated by the exactly opposite behavior of the compensationelement.

[0048] Naturally further objects of the invention are disclosedthroughout other areas of the specification and claims.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0049]FIG. 1 shows a generalized flow cytometer.

[0050]FIG. 2 shows a second view of a generalized flow cytometer.

[0051]FIG. 3 shows a comparison of univariate histograms from flowcytometers (#1, #2, and #3) without the amplifier invention (FIG. 3A)with univariate histograms for the same flow cytometers using aparticular embodiment of the amplification invention (FIG. 3B)illustrating the improved resolution between X-chromosome bearing andY-chromosome bearing populations of bovine spermatozoa.

[0052]FIG. 4 shows univariate and bivariate histograms illustrating theconventional resolution between X-chromosome bearing and Y-chromosomebearing populations of bovine spermatozoa.

[0053]FIG. 5 shows univariate and bivariate histograms illustratingimproved resolution between X-chromosome bearing and Y-chromosomebearing populations of bovine spermatozoa using a particular embodimentof the amplification invention.

[0054]FIG. 6 shows a second example of univariate and bivariatehistograms illustrating the conventional resolution between X-chromosomebearing and Y-chromosome bearing populations of bovine spermatozoa.

[0055]FIG. 7 shows a second example of univariate and bivariatehistograms illustrating the improved resolution between X-chromosomebearing and Y-chromosome bearing populations of bovine spermatozoa usinga particular embodiment of the amplification invention.

[0056]FIG. 8 shows univariate and bivariate histograms illustrating theconventional resolution between X-chromosome bearing and Y-chromosomebearing populations of equine spermatozoa.

[0057]FIG. 9 shows univariate and bivariate histograms illustrating theimproved resolution between X-chromosome bearing and Y-chromosomebearing populations of equine spermatozoa using a particular embodimentof the amplification invention.

[0058]FIG. 10 shows univariate and bivariate histograms illustrating theimproved resolution between X-chromosome bearing and Y-chromosomebearing populations of equine spermatozoa nuclei using a particularembodiment of the amplification invention.

[0059]FIG. 11 shows a particular embodiment of the circuit boardmodification to make the amplification invention with respect to aMoFlo® flow cytometer.

[0060]FIG. 12 shows an electrical schematic diagram of a particularembodiment of the amplification invention with respect to a MoFlo® flowcytometer.

[0061]FIG. 13 shows the laser beam pattern using conventional beam shapeoptics (FIG. 13A) and the laser beam pattern using the reduced heightbeam shape optics (FIG. 133B).

[0062]FIG. 14 shows a bar graph that compares the purity of separatedX-chromosome bearing spermatozoa (FIG. 14A) and Y-chromosome bearingspermatozoa (FIG. 14B) using conventional technology or using theamplification invention independently or in conjunction with reducedheight beam shaping optics.

[0063]FIG. 15 shows a front view of the reduced height beam shapingoptics.

[0064]FIG. 16 shows a top view of the reduced height beam shapingoptics.

[0065]FIG. 17 shows a perspective and two cross sections of the objectorienting nozzle invention.

[0066]FIG. 18 shows a graded series of cross sections of the objectorienting nozzle invention.

[0067]FIG. 19 shows a front view and an end view of an embodiment of thebeveled injection tube invention.

[0068]FIG. 20 illustrates the removal of undesired unorientedspermatozoa (RUUS) invention by comparison of signal(s) from theoriented spermatozoa (FIGS. 20A and 20B) and the signal(s) from theunoriented spermatozoa (FIGS. 20C and 20D).

[0069]FIG. 21 shows a perspective of another embodiment of the beveledinjection tube invention having a paddle shaped beveled blade.

[0070]FIG. 22 shows a conventional optics technology coupled to a flowcytometer.

[0071]FIG. 23A shows the shape and size of a typical spermatozoon andFIG. 23B shows the difference between correctly and non-correctlyorientated spermatozoa.

[0072]FIG. 24 shows an embodiment of the invention having constructionallowing the measurement of two signals, for example volume andorientation.

[0073]FIGS. 25A and B shows an embodiment of the invention having twohalves with a pinhole corresponding to each half, FIG. 25C shows animage plane of an embodiment of the invention, FIG. 25D shows anembodiment of the invention having two independently rotatablepolarizers.

[0074]FIGS. 26A and 26B illustrates the compensation method for thefluid stream for an embodiment of the invention, FIG. 26C shows anembodiment of a compensation element, 26D shows another embodiment of acompensation element where images of a fluid stream and from thecompensation element fall on top of each other in the image plane.

[0075]FIG. 27 shows an embodiment of the interference optics invention.

[0076]FIG. 28 shows an a second view of the interference opticsinvention.

V. MODE(S) FOR CARRYING OUT THE INVENTION

[0077] The invention involves isolated high purity X-chromosome bearingand Y-chromosome bearing populations of spermatozoa or sperm cells. Highpurity X-chromosome bearing and Y-chromosome bearing populations ofspermatozoa can comprise populations of intact live spermatozoa, and mayalso comprise populations of tailless spermatozoa (sperm nuclei), orpopulations of other viable or non-viable forms of spermatozoa, as maybe desired. While particular examples are provided that describe theinvention in the context of separating intact live sperm cells eachhaving a sperm cell head, necks, and tail, it should be understood thatthe technologies described can have various applications with respect tosperm nuclei as well. X-chromosome bearing and Y-chromosome bearingpopulations of spermatozoa should further be understood to encompassspermatozoa from any male of a species of mammal including, but notlimited to, spermatozoa from humans and spermatozoa from commonly knownanimals such as bovids, equids, ovids, canids, felids, goats, or swine,as well as less commonly known animals such as elephants, zebra, camels,or kudu. This list of animals is intended to be exemplary of the greatvariety of animals from which spermatozoa can be routinely sorted at 90%or greater purity, and is not intended to limit the description of theinvention to the spermatozoa from any particular species of mammals.

[0078] High purity separated spermatozoa from the various species ofmammals can be incorporated into products that can be used withartificial insemination protocols or as part of commercial businessmethods such as those as described in U.S. Patent Application Nos.60/211,093, 60/224,050, or Patent Cooperation Treaty Application No.US99/17165; or be used with low dose insemination protocols as describedin Patent Cooperation Treaty Application No. US98/27909, or used invitro fertilization of oocytes from animals, including humans, asdescribed in U.S. Patent Application No. 60/253,785, each of theabove-mentioned references are hereby incorporated by reference.

[0079] The use of the term purity or high purity should be understood tobe the percent of the isolated spermatozoa population bearing aparticular differentiating characteristic or desired combination ofcharacteristics. For example, where a population of spermatozoa areseparated based upon bearing an X-chromosome as opposed to aY-chromosome, a X-chromosome bearing population having 90% puritycomprises a population of spermatozoa of which 90% of the individualspermatozoa bear an X-chromosome while 10% of such population ofspermatozoa may bear a Y-chromosome. As such, high purity with respectto X-chromosome bearing populations or Y-chromosome bearing populationscan comprise a purity selected from the group consisting of between 90%to about 100%, between about 91% to about 100%, between about 92% toabout 100%, between about 93% to about 100%, between about 94% to about100%, between about 95% to about 100%, between about 96% to about 100%,between about 97% to about 100%, between about 98% to about 100%,between about 99% to about 100%.

[0080] Importantly, while numerous embodiments of the invention describeisolated high purity X-chromosome and Y-chromosome bearing populationsof spermatozoa, and while the description further discloses high purityspermatozoa separation devices and methods of how to isolate and how touse isolated high purity populations of spermatozoa, the basic conceptsof the invention should be understood to be applicable to other types ofparticles or events having particle differentiation characteristics orevent differentiation characteristics. It should be understood that theinvention can be applicable to a variety of circumstances in whichresolving small differences in photogenerated signal may be necessary,such as product defect detection, field flow fractionation, liquidchromatography, electrophoresis, computer tomography, gamma cameras,time of flight instruments, or the like as would be readily understoodby those skilled in those arts.

[0081] Moreover, while this disclosure provides descriptions ofembodiments of apparatus and methods for flow separation of X-chromosomebearing spermatozoa from Y-chromosome bearing spermatozoa, thedescription of these embodiments of the invention is not meant to reducethe scope of the invention to only flow separation of spermatozoa oronly to high purity flow cytometer spermatozoa separation systems butrather these examples are intended to exemplify the basic concepts ofthe invention in a practical manner so that they may be applied to thewide variety of applications.

[0082] Now referring to FIGS. 1 and 2, a flow cytometer embodiment ofthe invention is shown which includes a particle or cell source (1)which acts to establish or supply particles or cells stained with atleast one fluorochrome for analysis. The particles or cells aredeposited within a nozzle (2) in a manner such that the particles orcells are introduced into a fluid stream or sheath fluid (3). The sheathfluid (3) is usually supplied by some sheath fluid source (4) so that asthe particle or cell source (1) supplies the particles or cells into thesheath fluid (4) they are concurrently fed through the nozzle (2).

[0083] In this manner it can be easily understood how the sheath fluid(3) forms a sheath fluid environment for the particles or cells. Sincethe various fluids are provided to the flow cytometer at some pressure,they flow out of nozzle (2) and exit at the nozzle orifice (5). Byproviding some type of oscillator (6) which may be very preciselycontrolled through an oscillator control (7), pressure waves may beestablished within the nozzle (2) and transmitted to the fluids exitingthe nozzle (2) at nozzle orifice (5). Since the oscillator (6) acts uponthe sheath fluid (3), the stream (8) exiting the nozzle orifice (5)eventually and regularly forms drops (9). Because the particles or cellsare surrounded by the fluid stream or sheath fluid environment, thedrops (9) may entrain within them individually isolated particles orcells, and can be sperm cells with respect to some embodiments of theinvention.

[0084] Since the drops (9) can entrain particles or cells, the flowcytometer can be used to separate particles, cells, sperm cells or thelike based upon particle or cell characteristics. This is accomplishedthrough a particle or cell sensing system (10). The particle or cellsensing system involves at least some type of detector or sensor (11)which responds to the particles or cells contained within fluid stream(8). The particle or cell sensing system (10) may cause an actiondepending upon the relative presence or relative absence of acharacteristic, such as fluorochrome bound to the particle or cell orthe DNA within the cell that may be excited by an irradiation sourcesuch as a laser exciter (12) generating an irradiation beam to which theparticle can be responsive. While each type of particle, cell, or thenuclear DNA of sperm cells may be stained with at least one type offluorochrome different amounts of fluorochrome bind to each individualparticle or cell based on the number of binding sites available to theparticular type of fluorochrome used. With respect to spermatozoa, theavailability of binding sites for Hoechst 33342 stain is dependant uponthe amount of DNA contained within each spermatozoa. BecauseX-chromosome bearing spermatozoa contain more DNA than Y-chromosomebearing spermatozoa, the X-chromosome bearing spermatozoa can bind agreater amount of fluorochrome than Y-chromosome bearing spermatozoa.Thus, by measuring the fluorescence emitted by the bound fluorochromeupon excitation, it is possible to differentiate between X-bearingspermatozoa and Y-bearing spermatozoa.

[0085] In order to achieve separation and isolation based upon particleor cell characteristics, emitted light can be received by sensor (11)and fed to some type of separation discrimination system or analyzer(13) coupled to a droplet charger which differentially charges eachdroplet (9) based upon the characteristics of the particle or cellcontained within that droplet (9). In this manner the separationdiscrimination system or analyzer (13) acts to permit the electrostaticdeflection plates (14) to deflect drops (9) based on whether or not theycontain the appropriate particle or cell.

[0086] As a result, the flow cytometer acts to separate the particle orcells (16) by causing them to be directed to one or more collectioncontainers (15). For example, when the analyzer differentiates spermcells based upon a sperm cell characteristic, the droplets entrainingX-chromosome bearing spermatozoa can be charged positively and thusdeflect in one direction, while the droplets entraining Y-chromosomebearing spermatozoa can be charged negatively and thus deflect the otherway, and the wasted stream (that is droplets that do not entrain aparticle or cell or entrain undesired or unsortable cells) can be leftuncharged and thus is collected in an undeflected stream into a suctiontube or the like as discussed in U.S. patent application Ser. No.09/001,394, hereby incorporated by reference herein. Naturally, numerousdeflection trajectories can be established and collected simultaneously.

[0087] To routinely separate particles, cells, sperm cells, orspermatozoa (intact, live, fixed, viable, non-viable, or nuclei) intohigh purity X-chromosome bearing and Y-chromosome bearing populations,the particle differentiation apparatus or methods used must provide highresolution of the differentiation characteristics that are used as thebasis of analysis and separation.

[0088] With respect to spermatozoa, differentiating between the lightemitted by the fluorochrome bound to the nuclear DNA of X-chromosomebearing sperm cells and the light emitted by the fluorochrome bound tothe nuclear DNA of Y-chromosome bearing sperm cells may be difficult asdiscussed above.

[0089] In many applications, the total emitted light from photoemissiveevents incident to the detector, which can be a photomultiplier tube,can be high while the difference between the emitted light of eachphotoemissive events to be differentiated can be small. The problem canbe exacerbated when the photoemissive events happen serially at highrate of speed and the time period between photoemissive events is short,such as with high speed cell sorting using flow cytometers. Whenseparating particles, cells, or sperm cells based upon the difference inbound fluorochrome the cells flow past an excitation source and a highnumber of emissive events per second can be established. As a result,the amount of emitted light generated in the stream of particles, cells,or sperm cells, can be enormous. As the speed of the stream isincreased, the intercept point with the excitation source becomes verybright. This high level of incident light upon the photocathode of thephotomultiplier tube can cause a very low signal to background signalratio. The amount of background signal can be further exacerbated whenfluorochrome such as Hoechst 33342 can be used to label the nuclear DNAof sperm cells.

[0090] Most solutions to the problem have focused on decreasing thetotal amount of light flux upon the photocathode tube by placing opticalfilters in front of the photomultiplier tube. This approach does notchange the proportion of signal to background signal and subsequentattempts to increase the sensitivity of the photomultiplier tubegenerates additional background signal as the photomultiplier tubesaturates from the amount of background signal.

[0091] Typically, photomultiplier tubes have an operation voltage rangeof about 400 volts to about 900 volts. The lower limit of linearoperation of standard photomultiplier tubes, such as the R928 and R1477photomultiplier tubes available from Hamamatsu Corporation, may be about300 volts. As such, equipment or instruments which employphotomultiplier tubes are configured to operate such photomultipliertubes at or above 400 volts. Even where reduction of the number ofelectrons at the anode is desired, as disclosed in U.S. Pat. Nos.4,501,366 and 5,880,457 the voltage between the photocathode and thefirst dynode is maintained at a high voltage and reduction of theelectrons at the anode is accomplished by either decreasing the voltageto the remaining dynodes, or the inherent dark noise or shot noise isfiltered out electronically.

[0092] Unexpectedly, reducing the amount of voltage to thephotomultiplier tube below 400 volts to about 280 volts, or about 250volts, or even to 0 volts can allow small differences in photoemissivelight to be differentiated even when the total light emitted from eachphotoemissive event is high, or even when there are a high number ofbright serial events per second. With respect to the rate ofphotoemmissive events generated from the irradiation of fluorochromesbound to the nuclear DNA of spermatozoa, the invention allows the rateof photoemmissive events that can be achieved during separation ofspermatozoa into X-chromosome bearing and Y-chromosome bearingpopulations to be increased to a separable event rate of at least 5000separable events per second, at least 6000 separable events per second,at least 7000 separable events per second, at least 8000 separableevents per second, at least 9000 separable events per second, at least10,000 separable events per second, at least 11,000 separable events persecond, at least 12,000 separable events per second, at least 13,000separable events per second, at least 14,000 separable events persecond, at least 15,000 separable events per second, at least 16, 000separable events per second, at least 17,000 separable events persecond, at least 18,000 separable events per second, at least 19,000separable events per second, at least 20,000 separable events persecond, at least 25,000 separable events per second, at least 30,000separable events per second, and at least 35,000 separable events persecond, or greater.

[0093] As a specific example, existing Cytomation SX MoFlo® sorting flowcytometers are configured to operate the photomultiplier tube at 400volts minimum. The gain can be adjusted to operate the photomultipliertube at higher voltages but not lower voltages. SX MoFlo® flowcytometers can be converted by reconfiguring the photomultipliercontrollers. The R16C resistor (2.49 kilohms) on channel three can bereplaced by a 2.0K resistor to alter the gain of the amplifier thatcontrols the photomultiplier tube. This conversion allowed thephotomultiplier tube to be operated at about 280 volts. Similarconversion of SX MoFlo® flow cytometers with two 3.75 kilohm resistorsin parallel, or a 1.3 kilohm resistors can allow the photomultipliertube to be operated at voltages of about 200 volts, or just above zerovolts, respectively. Also with respect to this conversion, the neutraldensity filter in front of the photocathode can also be removed as aresult of operating the photomultiplier tube outside of the typicaloperation voltage range.

[0094] This conversion unexpectedly increases the signal to noise ratioof the photoemissive event as it is translated to an electronic signalby the photomultiplier tube. The cleaner signal may then be amplified byincreasing the gain amplifier to the analog to digital converter of theanalyzer (13) to the appropriate level and output may be generated asunivariate or bivariate histograms.

[0095] Now referring to FIG. 3, a comparison of univariate histogramsgenerated on three different SX MoFlo® flow cytometers (#1, #2, #3)prior to the use of the invention (FIG. 1A), and using the invention(FIG. 1B) with respect to the separation of intact live ejaculatedbovine sperm are shown. As can be understood from the univariatehistograms, the resolution (the apparent differentiation of theX-chromosome bearing population from the Y-chromosome bearing populationrepresented by the valley between peaks) of intact live X-chromosomebearing spermatozoa (17) from live Y-chromosome bearing spermatozoa (18)can be substantially improved by use of the invention.

[0096] The mean separation rate or sort rates of intact live spermatozoaprior to use of this embodiment of the invention with the SX MoFlo® flowcytometers was about 17.9×10⁶/4.5 hours of both X-chromosome bearingspermatozoa and Y-chromosome bearing spermatozoa (i.e. about 1,100separations or sorts per second in each of two streams—the first streamX-chromosome bearing spermatozoa and the second stream Y-chromosomebearing spermatozoa) at about 87% purity with a range of 84% to 93%purity. The separable event rate was 22,000, 23,000, and 20,000respectively for the three sorts.

[0097] The mean sort rates of live spermatozoa after the above-mentionedconversion was about 40.3×10⁶/4.5 hour sort (i.e. about 2,500 sorts persecond per stream) at about 90.8% purity with a range of 89% to about92%. The events per second were 13,000, 15,000, and 19,500 respectivelyfor the three sorts.

[0098] As can be understood from the data not only did this embodimentof the invention result in increased purity of the separated spermatozoapopulations but also allowed the separation rate or sort rate to be morethan doubled while the separable events rate was actually decreased.

[0099] Similarly, referring now to FIGS. 4 and 5, which show bivariatehistograms from sorting of intact live bull spermatozoa with the SXMoFlo® flow cytometer #1 prior to using the invention (FIG. 4) and afterthe above-mentioned conversion (FIG. 5). Prior to using the invention,the SX MoFlo® flow cytometer was initially operated at 440 volts at thephotocathode with the laser adjusted to 135 MW, a gain of 1× and with aneutral density filter of 1.0 ({fraction (1/10)}th amplitude) at about10,000 events per second. Upon using the invention, the SX MoFlo® flowcytometer was operated at about 262 volts at the photocathode, with thelaser adjusted to about 100 mW, a gain of 4×, without the neutraldensity filter, at about 10,000 separable events per second. As can beunderstood from this data there is a large increase in resolution asevidenced by the increased depth of the valley between the X-chromosomebearing population (19) and the Y-chromosome bearing population (20).

[0100] Similarly, referring now to FIGS. 6 and 7, which show bivariatehistograms from sorting of intact live bull spermatozoa with the SXMoFlo® flow cytometer #2 before using this embodiment of the invention(FIG. 6) and upon using this embodiment of the invention (FIG. 7)operated at the same parameters as shown in FIGS. 3 and 4 respectively.Again, there can be a large increase in resolution as evidenced by thedepth of the valley between the X-chromosome bearing population (21) andthe Y-chromosome bearing population (22).

[0101] Now referring to FIGS. 8 and 9, which show bivariate histogramsfrom separation or sorting of intact live equine spermatozoa with the SXMoFlo® flow cytometer before using this embodiment of the invention(FIG. 8) and upon using this embodiment of the invention (FIG. 9). Whenusing this embodiment of the invention, live equine spermatozoa wereseparated or sorted with the laser power at 100 mW with thephotomultiplier tube voltage below 300 volts. The separation rates orsort rates exceeded 4,800 sorts per second average at 12,000 events persecond. The increased resolution of the X-chromosome bearing population(23) and the Y-chromosome bearing population (24) is dramatic. The datashows that about 8 to about 9 channels separation can be achieved withthis embodiment of the invention as compared to 5 channels of separationbetween the peaks without the use of this embodiment of the invention.The purity of both the sorted X-chromosome bearing population and thesorted Y-chromosome bearing population was about 93%.

[0102] Now referring to FIG. 10, which shows a univariate histogram anda bivariate dot plot from sorting of Hoechst 33342 stained stallionsperm nuclei (S-05400) separated using this embodiment of the invention.The nuclei were prepared from freshly ejaculated stallion sperm. Thesperm were washed by centrifugation, sonicated and the resultant headsand tails separated using Percoll density gradient centrifugation. theisolated heads were washed, fixed with 2% formalin and then stained withHoechst 33342. The stained nuclei were stabilized using sodium azide(0.5%). The sample was run at 5000 events per second to produce thehistograms. The stained nuclei were then used to calibrate an SX MoFlo®flow cytometer was converted as above-mentioned to incorporate thephotomultiplier tube embodiment of the invention. Compensation was usedto level the two populations (X stained nuclei and Y stained nuclei) inthe bivariate plot. Note that the two populations of equine sperm nucleiare nearly fully resolved to baseline as shown by the univariate plot.

[0103] Now referring to FIG. 11, a modification specifically for SXMoFlo® flow cytometer includes the use of two resistors in parallel toprovide the correct value of 1.8K. Two 3.57K resistors (25) and (26) areequal to about 1.785K which can be sufficiently close to the value to beeffective. With this modification the photomultiplier tube on thisparticular instrument can then be run at about 200 volts. Naturally, asimilar modification can be made to other flow cytometer instruments orother instruments which use a photomultiplier tube to measure the amountof light emitted from particular events. FIG. 12, provides a electricalschematic diagram for this particular embodiment of the invention.

[0104] Another important embodiment of the invention can be a reducedheight irradiation beam pattern optics. As shown by FIG. 13A,conventional irradiation beam shaping optics generate a beam pattern(27) that can have a height can be greater than much greater than theheight of the sperm cell head(s) (28) passing through it. As a result,more than a single sperm cell head containing fluorochrome bound DNA canenter the irradiation beam pattern at the same time. In that case, thefluorochrome(s) bound to the DNAs contained within the multiple spermheads can be excited simultaneously and fluoresce within a singleemissive event. As such, the prior or subsequent emissive event caninclude coincident light flux contributed from other sperm head(s) inthe beam pattern (27). This results in a reduced difference in meanlight flux between light emissive events which distinguish betweenX-chromosome bearing spermatozoa and Y-chromosome bearing spermatozoa.It can also decrease the difference in mean light flux between eventsthat compare light emissions of X-chromosome bearing spermatozoa orY-chromosome bearing spermatozoa. Importantly, coincident excitation offluorochrome bound to multiple DNAs increases the mean brightness of theevents making the measurable difference in light flux between events aneven smaller percentage of the total light flux emitted. This makesquantification of the differences between events even more difficult.

[0105] By reducing the height of the beam shape as shown by FIG. 13B,the coincidence of multiple sperm heads being within the reduced heightbeam (29) pattern during the same measured event is reduced. Thisresults in an increased mean difference between light emissive eventswhich distinguish between X-chromosome bearing spermatozoa andY-chromosome bearing spermatozoa. It can also reduce the mean totallight flux for each measured emissive event. For particular embodimentsof the invention used for sorting bovine sperm which have a nucleus ofabout 9 μm, it has been found that the height of the beam can be about20 μm. In this application, it has been found that vertical beam heightsof less than 20 μm did not provide an additional gain in resolution.

[0106] Referring to FIG. 14, it can be understood that the use ofreduced height irradiation beam pattern optics can improve the purity ofsorted populations of X-chromosome bearing bovine spermatozoa (FIG. 14A)and sorted populations of Y-chromosome bearing bovine sperm (FIG. 14B)that have been stained with Hoechst 33342 stain. This is true for both25% and 40% sort gates of the univariate peak. As can be furtherunderstood from FIG. 14, the reduced height beam pattern optics canimprove purity of separated spermatozoa independent of any other aspectof the invention, such as modification of photomultiplier circuitryembodiment of the invention (new PMT) as described above, or can be usedin conjunction with the modified photomultiplier embodiment of theinvention to increase the purity of separated spermatozoa samples evenfurther.

[0107] Another advantage of the reduced height beam pattern optics canbe that the transit time of the spermatozoa in the excitation laser beamor irradiation beam can be reduced. A reduced amount of irradiation timewithin the excitation laser beam may result in less stress or damage tothe spermatozoa.

[0108] Again referring to FIG. 14B, it can be understood that thereduced height beam pattern can be used in conjunction with airradiation beam pattern having greater area than conventionally used.For example, conventional beam patterns (27), such as that shown in FIG.14A, have an elliptical pattern of about 30 um×80 um while the inventionwhen used for sorting bovine sperm generates optimal resolution betweenX-chromosome bearing and Y-chromosome bearing populations when the beamhas a 20 um×160 um beam pattern (29). The 20 um×160 um beam pattern hasapproximately 1.3 times the area of the 30 um X 80 um beam pattern. Assuch, there can be an inverse proportion in loss of energy at theincident point. This makes it possible to increase the excitation laserpower without concern for increasing the irradiation damage to thespermatozoa. For example, if an instrument has conventional beam shapingoptics that produce a 30 um×80 um irradiation beam pattern and theexcitation laser is conventionally powered at 150 mW, then particularembodiments of the invention with a 20 um×160 um beam pattern can havean excitation laser powered at 300 mW without increasing the totalamount of power at the incident point. Alternately, the excitation lasercan be run at 150 mW to take advantage of the lower per unit areairradiation energy, decreased irradiation damage, longer laser life, andthe like.

[0109] In comparison to conventional beam shaping optics andconventional photomultiplier tube amplification devices, the reducedheight beam pattern optics invention and the photomultiplier tubeamplification invention can increase the purity of X-chromosome bearingand Y-chromosome bearing populations of spermatozoa by about 4%, ormore.

[0110] The beam shaping optics invention (30) can be installed to a flowcytometer as shown in FIGS. 15 and 16. As can be understood, the lightemitted (31) by laser excitation of fluorochrome(s) bound to the DNAcontained within spermatozoa can be detected by photomultiplier tubes(32) situated at 0 and 90 degrees relative to the flat surface of thesperm head (28) as it flows through the excitation laser beam pattern.

[0111] As can be understood, stained spermatozoa must be pumped throughthe excitation beam or irradiation beam in a precise manner so that eachsperm head is oriented with the flat surface of the sperm head directedtoward the photomultiplier tube that is the 0 degree detector. Accuratemeasurement of the DNA content of the spermatozoa can only be measuredfrom the flat surface of the paddle-shaped sperm head (28). Thus, onlythat proportion of the spermatozoa that enter the excitation beam in theproper orientation can be measured accurately and sorted based upon DNAcontent.

[0112] Now referring to FIGS. 17, 18, and 19, particular embodiments ofthe invention can also have an particle or sperm cell orienting nozzle(33) that hydrodynamically forces the flattened sperm head into theproper orientation as they pass in front of the photomultiplier(s). Asshown by FIG. 17, the orienting nozzle has interior surfaces (34) thatform a cone-like shape. The internal cone gradually changes fromcircular at the inlet end (35) into a highly elliptical shape near theorifice (36) where the stream exits the tip. The orifice (36) can becircular rather than elliptical. Thus, the internal aspect of theorienting nozzle (34) goes from a round entrance to a narrow ellipse toa round exit shortly before the orifice (36). This internal shape isfurther clarified by the cross sections of the orienting nozzle shown byFIG. 18.

[0113] As shown by FIGS. 19 and 21, the injection tube (37) (which maybe about 0.061 inches in diameter) can be used with the orientationnozzle (or with a conventional nozzle) (33) which can be beveled nearthe tip to form a blade (38). The flattened blade (38) can be orientedat an angle 90 degrees from the greatest dimension of the ellipse in theorientation nozzle (33). The internal diameter of the injection needlecan be about 0.010 inch in diameter forming a rounded orifice (39) inthe center of the flattened needle tube blade (38).

[0114] In particular embodiments of the beveled injection tube thebeveled blade can be configured in the paddle shape illustrated by FIG.21. The paddle shaped beveled blade can assist in maintaining laminarflow of the sheath fluid within the nozzle (whether conventional nozzleor orienting nozzle). As such, the laminar flow of liquid maintained bythe paddle shaped beveled blade presents less disruption of the objectsinjected into it. Spermatozoa introduced into the laminar flow of sheathfluid maintained by an embodiment of the injector tube invention havingthe paddle shaped beveled blade allows for a 20%, 30%, 40%, 50% or evengreater increase in spermatozoa sorting rates over conventionalinjection tube technology. High speed sorting of spermatozoa at rates ofabout 4,000 to about 10,000 sorts of each sex per second can beaccomplished. High purity (90% or greater) of the X-chromosome bearingand Y-chromosome bearing populations can be established at even thesehigh sort rates. The injector tube invention with the beveled paddleshaped tip can be used independently of or in combination with the otherinventions described herein or other technology such as that describedin U.S. patent application Ser. No. 09/454,488 or International PatentApplication No. PCT/US00/42350, each hereby incorporated by reference.

[0115] As shown by FIG. 21, certain embodiments of the beveled bladeinjector tube invention or beveled blade paddle shape invention canfurther include laminar flow enhancement grooves (40). The laminar flowenhancement grooves (40) assist in maintaining a laminar flow to theorifice of the injector tube. Again, the enhanced laminar flow allowsfor more spermatozoa to maintain the correct orientation in the laminarsheath fluid flow resulting in higher numbers of sortable event rateswhich in turn leads to higher sort rates for each sex or spermatozoa.

[0116] In another embodiment of the invention, the orienting nozzleorifice (39) or other conventional can be sized to form droplets whichencapsulate intact live sperm as they exit the orifice (39).Encapsulation of the sperm cells does not occur in conventional spermcell entrainment technology. Rather a portion of the sperm cell tailresides outside of the droplet. For example, bovine sperm cells have alength of about 13.5 microseconds when the fluid stream has a pressureof about 50 pounds per square inch (i.e. the length of time for theentire length of the sperm cell to pass through the irradiation beam atabout 50 pounds per square inch fluid stream pressure). Conventionaldroplet formation techniques for entraining bovine sperm cells establisha 14 microsecond droplet (i.e. the time it takes to form a singledroplet waveform in a fluid stream) from a nozzle having an orifice witha diameter of about 70 micrometers which can be made responsive to anoscillator operated at about 35 kilohertz. As such, a portion of thesperm cell tail readily protrudes from the droplet. To prevent the spermcell tail from protruding from the droplet, one embodiment of thedroplet encapsulation invention provides an orifice of about 100micrometers that can produce a droplet of about 28 microseconds at about50 pounds per square inch at about 30 kilohertz. By entirelyencapsulating the intact live sperm cell, including the tail portion,the sperm cell interacts with the nozzle less upon charging of thedroplet and the deflection of the droplet can be more accurate. Thisleads to less cross contamination of X-chromosome bearing sperm withY-chromosome bearing sperm and also allows deflected spermatozoa to bemore uniformly collected. Sperm that are uniformly deflected can bedirected to collection surfaces that are cushioned by various liquids.Cushioning the separated spermatozoa can be important in reducing stressas described in U.S. patent application Ser. No. 09/001,394, herebyincorporated by reference. With respect to spermatozoa from otherspecies of mammals, the invention can be varied to produce droplet sizesto encapsulate the varying lengths of sperm cells. Depending on thelength of the spermatozoa and the pressure of the fluid stream thedroplet encapsulation invention can still achieve droplet formationrates of at least 10,000 droplets per second, at least 20,000 dropletsper second, at least 30,000 droplets per second, at least 40,000droplets per second, at least 50,000 droplets per second, at least60,000 droplets per second, at least 70,000 droplets per second, atleast 80,000 droplets per second, at least 90,000 droplets per second,at least 100,000 droplets per second and so on up to about 200,000droplets per second in some embodiments of the droplet encapsulationinvention.

[0117] Even with the orienting nozzle invention there will be a certainnumber of spermatozoa, or particles, which are not properly oriented inthe beam pattern. As described above, if the orientation of a sperm headis not proper then the DNA content cannot be measured accurately basedupon the emitted light. Particular embodiments of the present inventionprovide for the removal of undesired unoriented spermatozoa (RUUS) orparticles within a fluid stream.

[0118] Referring now to FIGS. 16 and 20A, it can be understood thataccurate measurement of the DNA content of a spermatozoa depends uponthe flat surface of the paddle-shaped sperm head (28) being orientedproperly with the detector. Thus, only that proportion of thespermatozoa that enter the excitation beam in the proper orientation asshown by FIGS. 16 and 20A can be measured accurately and sorted in toX-chromosome bearing and Y-chromosome bearing populations based upon DNAcontent. As shown by FIGS. 20A and 20B, spermatozoa which transitthrough the excitation beam in proper orientation generate an orientedemission signal plot (40) that can be shaped differently than theunoriented emission signal plot (41) that is generated by unorientedspermatozoa shown by FIG. 20D. Naturally, the shape of the unorientedemission signal plot (41) generated by unoriented spermatozoa will varydepending on the degree of improper orientation in the excitation beam.These improper orientations can include the orientation shown in FIG.20C but can also include all manner of orientations that rotate thesperm head any portion of a rotation that orients the surface of thepaddle-shaped head out of alignment with the detector (proper alignmentshown by FIG. 16), or any portion of a rotation that orients the axis ofthe sperm head (42) out of alignment with the direction of flow.Naturally, proper orientation may be defined differently from species tospecies. For some species, in which the sperm head is not paddle-shaped,the proper orientation within the excitation beam, or relative to thedetectors or otherwise, may be defined by other anatomicalcharacteristics or signal characteristics. Nonetheless, an optimizedsignal for the properly oriented spermatozoa of various species withinthe excitation window can be generated as the standard emission signalplots for subsequent comparison with serial emission events.

[0119] By comparing the shape (or the integrated area or both) of eachemission signal plot with the standard emission signal plot (or standardintegrated area or both) established for an oriented spermatozoa of aspecies of mammal, unoriented sperm can be identified, the signalsubtracted from univariate or bivariate histograms, and the unorientedsperm can be affirmatively removed, if desired, so that unoriented spermare not collected into either the X-chromosome bearing population or theY-chromosome bearing population.

[0120] Importantly, as the invention(s) improve(s) resolution betweenthe two spermatozoa populations being separated which increase the rateat which the populations can be separated from one another, and improvesthe purity of the populations that are separated. As such, it is nowpossible to sort spermatozoa at remarkably high speeds. Sortable orseparable event rates can be as high as about 35,000 per second (notincluding coincident events—multiple spermatozoa within theexcitation/detection window at the same time). Sortable or separableevent rates correlate with high separation or sort rates which can beabout 5000 to about 11,000 intact live sperm of each sex per second witha purity of 90%, 92%, 93%, 95%, or greater. The above-describedinventions also allow for even higher purity X-chromosome bearing andY-chromosome bearing populations to be obtained of about 97% to about98% or even higher by reducing the sort or separation rates to around2000 live sperm of each sex per second.

[0121] As can be understood, the above inventions described areparticularly important in achieving the highest possible sortable orseparable event rates and highest possible resulting separation rateswhich can be at least 1,000 separations per second, at least 2,000separations per second, at least 3,000 separations per second, at least4,000 separations per second, at least 5,000 separations per second, atleast 6,000 separations per second, at least 7,000 separations persecond, at least 8,000 separations per second, at least 9,000separations per second, or at least 10,000 separations per second ofeach sex per second, or greater.

[0122] The invention allows for high speed sorting, as set forth above,of spermatozoa even when they are difficult to stain, or have otheranatomical or chemical features, that make differentiation between theX-bearing chromosome and Y-bearing chromosome populations moredifficult. Even in these difficult cases, high purity X-chromosomebearing and Y-chromosome bearing populations of bovine spermatozoa canbe isolated at high purity of 92% to 93% by achieving sortable eventrates of about 15,000-20,000 sortable events per second or higher asdescribed above, and sort or separation rates of intact live spermatozoaof each sex (X-chromosome bearing and Y-chromosome bearing) of 2000intact live sperm of each sex per second.

[0123] Now referring to FIGS. 23 and 24, an embodiment of the inventionutilizes differential interference contrast technology to measure thevolume of a particle or capsule. A basic embodiment of the invention cancomprise particles that have a difference volume, such as sperm cellheads (28) that have a difference in volume between X-chromosome bearingand Y-chromosome bearing sperm cells. An electromagnetic radiationsource (43) generates electromagnetic radiation or a beam ofelectromagnetic radiation (44) having initial waveform characteristicsdifferentially responsive to the difference in volume between theparticles or sperm cell heads (28). The electromagnetic radiation whichcan be laser light, but could also be numerous types of electromagneticradiation including, but not limited to, microwave radiation,ultraviolet radiation, or the like. Upon traversing the particle orcapsule or sperm head volume containing phase shifting material theelectromagnetic radiation can be focused through an objective lens (45)onto a detector (46) responsive to the waveform characteristics of theelectromagnetic radiation. The detector can be coupled to an analyzer(47). The analyzer can differentiate between particles based on thechange in the waveform characteristics prior to traversing the volume ofthe particle and after traversing the volume of the particle and cananalyze the signal based on integrated areas or signal shape or both. Incertain embodiments the invention analyzing waveform characteristics cancomprise superimposing initial waveform characteristics with alteredwaveform characteristics upon traversing the volume of the particle,capsule, or sperm cell head. Superimposing the initial waveformcharacteristics and the phase shifted waveform characteristic candifferentially modulate the intensity of the beam of electromagneticradiation in manner that correlates to the amount of phase shift mediathe electromagnetic radiation traverses. The invention may also includeadditional filters (48), such as color filters.

[0124] Now referring to FIG. 24, an embodiment of the optics inventioninvolves using differential interference contrast optics that increasethe actual distance over which the light is split up compared toconventional DIC microscopy which corresponds to the resolution limit ofthe microscope. In this embodiment of the invention, the induced splitis larger than the size of the objects, thus giving rise to twoindividual images, separated laterally, originating from one object. Thesecond modification involves using plates of birefringent material, suchas Savart plates, at a location away from the objective lens. Thisembodiment of the invention is easier to construct since thebirefringent materials do not have to be located inside the objectivehousing. In conventional DIC microscopes the birefringent material isused in the form of so-called Wollaston prisms, that have to be locatedinside the objective housing, making it necessary to use expensiveobjective lenses that have been manufactured specifically for thispurpose.

[0125] Components of an embodiment of the invention may be arranged inline with each other and consist of: a source of electromagneticradiation (43), for example, a Mercury arc lamp; a spectral adjustmentelement, for example, a bandpass filter; a polarization adjustmentelement (49), for example, a sheet polarizer (53) and a waveplate (54)responsive to a rotatable mount; a light condenser (51) allowing thelight to be condensed onto the particle or sperm cell, for example, acondenser lens, or set of lenses, or microscope objective; a fluidstream (8) that may contain particles or sperm cells (28), for example afluid jet ejected under pressure; a light collector (45) to collect thelight from the particle or cell, for example a 50×high working distancemicroscope objective and a tube lens; a beam splitter (50) to split upthe beam into two, or more, components, for example, a piece ofbirefringent material in the form of a Savart plate, mounted in such away that its orientation and location can be controlled accurately;image light selector (55) to select only the light corresponding to theparticle or sperm cell, for instance a set of pinholes, one pinhole (53)for each of the images formed.

[0126] In one embodiment of the invention, the components may bearranged in such a way that the light source (43) or its image arelocated at the back focal plane of the light condenser (45) oftenreferred to as Köhler type illumination. The image of the object planemay best coincide with the object light selector (55) or pinhole(s)(53), in order to capture the light from individual particles or spermcells. As shown in FIGS. 27 and 28, components can be mounted on asturdy optical table, or bench, using mountings, posts, and holders.Components can be mounted in such a way that focusing of the objectplane can be done accurately. This can be done by equipping the fluidstream with a stream position controller, such as micrometers, in orderto turn the stream in and out of focus. In addition it may be necessaryto equip the light condenser (51) with a light condenser positioncontroller (61) allowing it to be focused onto the object plane. It maybe necessary to take special care about the mounting of the birefringentelements or beam splitter (50), a three axis rotation element may bepreferable.

[0127] Now referring to FIG. 25, embodiments of the present inventionmay also include the use of both generated images, in order to determinethe orientation of a asymmetrical particles the fluid stream, including,but not limited to, spermatozoa such as bull sperm cells. An orientationassessment embodiments of the invention can include an optical systemthat allows for control of the polarization state of the light enteringthe system for both generated images independently. The interferenceoptics invention may further provide polarization adjustment element(56) that controls the polarization state of light entering the system.For the orientation detection invention the polarization adjustmentelement (56) may be selected in such a way that it consists of twoparts, that are imaged onto image light selector (55) that in oneembodiment of the invention contains the pinholes (53). This can beaccomplished by locating the polarization adjustment element (56) in theconjugate plane of the image plane (55), or by using other optics, toaccomplish the same thing. A simple example of this component may be a‘half-shade’ piece, for instance consisting of two hemi-circular partsof polarizing material, such as sheet-polarizer, the orientation angleof which may be chosen independently. Each pinhole in the image planecan fall in one of the halves of said hemisphere. The polarizationangles can be chosen in such a way that the signal of one pinhole (53)corresponds to the volume, and is relatively independent from theorientation angle of the passing object, and the other pinhole (53) hasa signal that depends, to a great degree, upon this orientation angle.The two signals may be processed by analyzer (47) in a manner similar toa conventional multi-channel flow cytometry, as but one example. Withrespect to this example, bivariate dotplots can be made, and also allowthe user to select windows on this plot.

[0128] An improvement of the ‘half-shade’ piece described above may bethe construction shown by FIG. 25D. The same said two hemisphericalparts are projected onto the image plane but the way they are generatedis different. A mirror (57) breaks up the light (44) into thehemi-circular parts, and recombines them back to back. Each of thehalves traverses a separate means to control its polarization state. Anadvantage of this embodiment is that the polarization angles can becontrolled continuously and independently, thus facilitating theadjustment of the set-up. Materials used in this embodiment can besupplied by standard optical supply firms, and can be mounted in theset-up using similar mounting materials as used for the interferometricoptics.

[0129] Now referring to FIG. 26, In order to correct for artifactsintroduced by having light pass through a non-flat region of transparentmaterial, such as a substantially cylindrical fluid stream but includingother geometries as well, embodiments of the present invention disclosethe incorporation of a component similar in shape to the non-flatregion, but opposite in terms of relative refractive indices. In thespecific case of a flow cytometer this shape approximates a cylinder. Tocorrect for artifacts introduced by the fact that the objects to beassessed are located within a cylindrical stream of water, is theincorporation of an optical component (58) which can be in the shape ofa transparent cylinder, located inside transparent material (59) of ahigher refractive index. It may be preferred that the image of thestream and of the compensation element fall on top of each other in theimage plane. This can be done by locating the compensation elementbetween the objective lens and the image plane, and by incorporatingauxiliary lenses.

[0130] An embodiment of the optical component (58) can be located withina thin slice of transparent material of higher refractive index (59),for instance glass, or perspex, with a cylindrical hole drilled acrossit. Perspex has the advantage that it can be easier to drill a roundchannel into it. The cylindrical hole may be filled by a transparentmaterial, the refractive index of which is lower than that of thesurrounding material. The difference in refractive index between thesubstance and the surrounding material can be the same as but oppositeto the difference in refractive index between the water in the streamand the surrounding air for certain applications. It may not benecessary to have the cylinder the same size as the stream of water, aslong as magnification by the lenses used, makes the resulting images inthe image plane the same size. In some applications, it may be desiredor necessary to adjust the refractive index difference to compensate forthis magnification. Manufacturing of such element out of perspex can bequite simple, and can be done by most mechanical workshops that haveexperience with machining perspex or the selected material. It may bemade in such dimensions that it fits in a standard optical mountinghardware, to facilitate incorporation into the optics.

[0131] Exactly matching the refractive indices may be difficult. Anembodiment of the invention that facilitates adjustment can be to makethe substance inside the perspex, or other selected material, atransparent refractive index fluid (58), as but one example, an organicoil, or mixture of oils that have a refractive index close to thedesired one. Due to the fact that the refractive index of most fluidschanges with temperature, much more so than solids, or glasses, it maybe possible to fine-tune the difference in refractive index bytemperature. This may be done by incorporating a temperature controller(60).

[0132] Optical component (58) of transparent fluids or refractive indexfluids can be supplied by chemical supply firms. These firms often havedata regarding the refractive index of their fluids readily available.Some firms even offer fluids that are specially made to serve asrefractive index fluids, and have a guaranteed and stable refractiveindex. Temperature controllers and thermostats are supplied by manyfirms. A practical way to apply heat to the refractive index fluid canbe to use a hollow mounting made of heat conducting material, a metal asbut one example, containing the refractive index fluid. Using aconventional immersion thermostat cycler, found in many laboratories,water can be pumped through the mounting, thus keeping the element at afixed and controllable temperature.

[0133] The discussion included in this PCT application is intended toserve as a basic description. The reader should be aware that thespecific discussion may not explicitly describe all embodimentspossible; many alternatives are implicit. It also may not fully explainthe generic nature of the invention and may not explicitly show how eachfeature or element can actually be representative of a broader functionor of a great variety of alternative or equivalent elements. Again,these are implicitly included in this disclosure. Where the invention isdescribed in functionally-oriented terminology, each aspect of thefunction is accomplished by a device, subroutine, or program. Apparatusclaims may not only be included for the devices described, but alsomethod or process claims may be included to address the functions theinvention and each element performs. Neither the description nor theterminology is intended to limit the scope of the claims which now beincluded.

[0134] Further, each of the various elements of the invention and claimsmay also be achieved in a variety of manners. This disclosure should beunderstood to encompass each such variation, be it a variation of anembodiment of any apparatus embodiment, a method or process embodiment,or even merely a variation of any element of these. Particularly, itshould be understood that as the disclosure relates to elements of theinvention, the words for each element may be expressed by equivalentapparatus terms or method terms—even if only the function or result isthe same. Such equivalent, broader, or even more generic terms should beconsidered to be encompassed in the description of each element oraction. Such terms can be substituted where desired to make explicit theimplicitly broad coverage to which this invention is entitled. As butone example, it should be understood that all actions may be expressedas a means for taking that action or as an element which causes thataction. Similarly, each physical element disclosed should be understoodto encompass a disclosure of the action which that physical elementfacilitates. Regarding this last aspect, as but one example, thedisclosure of a “droplet separator” should be understood to encompassdisclosure of the act of “separating droplets”—whether explicitlydiscussed or not—and, conversely, were there only disclosure of the actof “converting liquid-gas”, such a disclosure should be understood toencompass disclosure of a “droplet separator” and even a means for“separating droplets”. Such changes and alternative terms are to beunderstood to be explicitly included in the description.

[0135] Additionally, the various combinations and permutations of allelements or applications can be created and presented. All can be doneto optimize the design or performance in a specific application.

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[0138] In addition, as to each term used it should be understood thatunless its utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood asincorporated for each term and all definitions, alternative terms, andsynonyms such as contained in the Random House Webster's UnabridgedDictionary, second edition are hereby incorporated by reference.However, as to each of the above, to the extent that such information orstatements incorporated by reference might be considered inconsistentwith the patenting of this/these invention(s) such statements areexpressly not to be considered as made by the applicant(s).

[0139] In addition, unless the context requires otherwise, it should beunderstood that the term “comprise” or variations such as “comprises” or“comprising”, are intended to imply the inclusion of a stated element orstep or group of elements or steps but not the exclusion of any otherelement or step or group of elements or steps. Such terms should beinterpreted in their most expansive form so as to afford the applicantthe broadest coverage legally permissible in countries such as Australiaand the like.

[0140] Thus, the applicant(s) should be understood to have support toclaim at least: I) each of the liquid to gas conversion devicesdescribed herein, ii) the related methods disclosed and described, iii)similar, equivalent, and even implicit variations of each of thesedevices and methods, iv) those alternative designs which accomplish eachof the functions shown as are disclosed and described, v) thosealternative designs and methods which accomplish each of the functionsshown as are implicit to accomplish that which is disclosed anddescribed, vi) each feature, component, and step shown as separate andindependent inventions, vii) the applications enhanced by the varioussystems or components disclosed, viii) the resulting products producedby such systems or components, ix) methods and apparatuses substantiallyas described hereinbefore and with reference to any of the accompanyingexamples, and the x) the various combinations and permutations of eachof the elements disclosed

[0141] In addition, unless the context requires otherwise, it should beunderstood that the term “comprise” or variations such as “comprises” or“comprising”, are intended to imply the inclusion of a stated element orstep or group of elements or steps but not the exclusion of any otherelement or step or group of elements or steps. Such terms should beinterpreted in their most expansive form so as to afford the applicantthe broadest coverage legally permissible in countries such as Australiaand the like.

[0142] The claims set forth in this specification by are herebyincorporated by reference as part of this description of the invention,and the applicant expressly reserves the right to use all of or aportion of such incorporated content of such claims as additionaldescription to support any of or all of the claims or any element orcomponent thereof, and the applicant further expressly reserves theright to move any portion of or all of the incorporated content of suchclaims or any element or component thereof from the description into theclaims or vice-versa as necessary to define the matter for whichprotection is sought by this application or by any subsequentcontinuation, division, or continuation-in-part application thereof, orto obtain any benefit of, reduction in fees pursuant to, or to complywith the patent laws, rules, or regulations of any country or treaty,and such content incorporated by reference shall survive during theentire pendency of this application including any subsequentcontinuation, division, or continuation-in-part application thereof orany reissue or extension thereon.

We claim:
 1. A method of isolating X-chromosome bearing sperm cells andY-chromosome bearing sperm cells, comprising the steps of: a. collectingsperm cells from a male of a species of mammal; b. determining a sexdifferentiation characteristic of said sperm cells; c. differentiatingbetween said sperm cells based upon said sex differentiationcharacteristic; d. separating differentiated sperm cells into aX-chromosome bearing population and a Y-chromosome bearing population;and e. producing separate X-chromosome bearing and Y-chromosome bearingpopulations of sperm cells each having a purity of greater than 90%. 2.A method of isolating X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells as described in claim 1, wherein said step ofdifferentiating between said sperm cells based upon said sexdifferentiation characteristic comprises assessing an amount of DNAwithin a nucleus of said sperm cells.
 3. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 1, wherein said step of differentiating between saidsperm cells based upon said sex differentiation characteristic comprisesassessing a volume of a capsule containing said amount of DNA.
 4. Amethod of isolating X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells as described in claim 3, wherein said step ofassessing said volume of said capsule containing said amount of DNAcomprises determining a volume of a nucleus within said sperm cells. 5.A method of isolating X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells as described in claim 3, wherein said step ofassessing said volume of a capsule containing said DNA comprisesdetermining the volume of a sperm cell head.
 6. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 5, wherein said step of assessing said volume of saidcapsule containing said amount of DNA further comprises the steps of a.generating a beam of electromagnetic radiation having initial wave formcharacteristics; b. traversing said volume of said capsule containingsaid amount of DNA with said beam of electromagnetic radiation; c.altering said initial wave form characteristics by traversing saidvolume of said capsule; and d. analyzing altered wave formcharacteristics.
 7. A method of isolating X-chromosome bearing spermcells and Y-chromosome bearing sperm cells as described in claim 6,wherein said step of altering said initial wave form characteristics bytraversing said volume of said capsule comprises shifting phase of saidinitial wave form of said beam of electromagnetic radiation.
 8. A methodof isolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells as described in claim 6, further comprising the step ofsuperimposing said beam of electromagnetic radiation having shiftedphase on said beam of electromagnetic radiation having said initial waveform characteristics.
 9. A method of isolating X-chromosome bearingsperm cells and Y-chromosome bearing sperm cells as described in claim8, further comprising the step of comparing electromagnetic radiationintensity of superimposed beams of electromagnetic radiation withintensity of said beam of electromagnetic radiation having initial waveform characteristics.
 10. A method of isolating X-chromosome bearingsperm cells and Y-chromosome bearing sperm cells as described in claim9, further comprising the step of determining said volume of saidcapsule containing said amount of DNA based upon a difference in saidlight intensity between said superimposed beams of electromagneticradiation and said beam of electromagnetic radiation having initialwaveform characteristics.
 11. A method of isolating X-chromosome bearingsperm cells and Y-chromosome bearing sperm cells as described in claim2, further comprising the steps of: a. introducing said sperm cells intoa fluid stream; b. analyzing said sperm cells entrained in said fluidstream; c. forming droplets a plurality having one of said sperm cellsentrained; d. charging each of said droplets differentially based uponsaid sex differentiation characteristic of said sperm cells entrained insaid droplets; e. deflecting each of said droplets; and f.differentially collecting each of said droplets based upon said sexdifferentiation characteristic of said sperm cells entrained in saiddroplets.
 12. A method of isolating X-chromosome bearing sperm cells andY-chromosome bearing sperm cells as described in claim 11, furthercomprising the step of maintaining said amount of DNA within saidnucleus of said sperm cell unstained.
 13. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 11, further comprising the step of staining saidamount of DNA within said nucleus of said sperm cell.
 14. A method ofisolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells as described in claim 13, further comprising the step ofirradiating stained DNA within said nucleus of said sperm cell.
 15. Amethod of isolating X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells as described in claim 14, further comprising thestep of detecting fluorescent light emitted from irradiated stained DNAwithin the nucleus of said sperm cell.
 16. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 15, wherein said step of detecting fluorescent lightfrom irradiated stained DNA within said nucleus of said sperm cellcomprises generating a signal with a photomultiplier tube.
 17. A methodof isolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells as described in claim 16, further comprising the step ofoperating said photomultiplier tube outside a typical operation voltagerange.
 18. A method of isolating X-chromosome bearing sperm cells andY-chromosome bearing sperm cells as described in claim 14, wherein saidstep of irradiating stained DNA within the nucleus of said sperm cellfurther comprises generating an irradiation beam pattern having areduced height.
 19. A method of isolating X-chromosome bearing spermcells and Y-chromosome bearing sperm cells as described in claim 18,wherein said reduced height comprises a height about equal to the lengthof said sperm cells along the longitudinal axis to about three times thelength of said sperm cells along the longitudinal axis.
 20. A method ofisolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells as described in claim 19, wherein said sperm cell has alongitudinal axis of about nine micrometers in length, and said heightof said irradiation beam pattern is about 20 micrometers.
 21. A methodof isolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells as described in claim 18, wherein said step of formingdroplets comprises forming droplets having sufficient size toencapsulate said sperm cell.
 22. A method of isolating X-chromosomebearing sperm cells and Y-chromosome bearing sperm cells as described inclaim 21, further comprising the step of ejecting said fluid stream froma nozzle having an orifice, wherein said orifice has a diameter of 70micrometers.
 23. A method of isolating X-chromosome bearing sperm cellsand Y-chromosome bearing sperm cells as described in claim 22, furthercomprising the steps of: a. orienting said sperm cells with respect to adetector; b. detecting light having characteristics differentiallyresponsive to sperm cell orientation to said detector; c. convertingsaid light having characteristics deferentially responsive to said spermcell orientation into at least one signal containing sperm cellorientation information; and d. determining orientation of said spermcell with respect to said detector.
 24. a method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 23, further comprising the step of differentiallycollecting said droplets based upon determined orientation of said spermcell with respect to said detector.
 25. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claims 1, 6, 12, 13, 17, 18, 21, or 23, wherein said purityof said X-chromosome bearing and said Y-chromosome bearing populationsof sperm cells is selected from the group consisting of between 90% toabout 100%, between about 91% to about 100%, between about 92% to about100%, between about 93% to about 100%, between about 94% to about 100%,between about 95% to about 100%, between about 96% to about 100%,between about 97% to about 100%, between about 98% to about 100%,between about 99% to about 100%.
 26. A method of isolating X-chromosomebearing sperm cells and Y-chromosome bearing sperm cells as described inclaims 1, 6, 12, 13, 17, 18, 21, or 23, further comprising the step ofestablishing a separable event rate from the group consisting of atleast 5000 separable events per second, at least 6000 separable eventsper second, at least 7000 separable events per second, at least 8000separable events per second, at least 9000 separable events per second,at least 10,000 separable events per second, at least 11,000 separableevents per second, at least 12,000 separable events per second, at least13,000 separable events per second, at least 14,000 separable events persecond, at least 15,000 separable events per second, at least 16, 000separable events per second, at least 17,000 separable events persecond, at least 18,000 separable events per second, at least 19,000separable events per second, at least 20,000 separable events persecond, at least 21,000 separable events per second.
 27. A method ofisolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells as described in claims 1, 6, 12, 13, 18, 21, or 23, whereinsaid step of separating said sperm cells comprises a separation rateselected from the group consisting of at least 500 separations persecond, at least 1,000 separations per second, at least 2,000separations per second, at least 3,000 separations per second, at least4,000 separations per second, at least 5,000 separations per second, atleast 6,000 separations per second, at least 7,000 separations persecond, at least 8,000 separations per second, at least 9,000separations per second, at least 10,000 separations per second.
 28. Amethod of isolating X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells as described in claims 1, 6, 12, 13, 18, 21, or 23,wherein said step of forming droplets each having one of said spermcells entrained comprises a droplet formation rate selected from thegroup consisting of at least 10,000 droplets per second, at least 20,000droplets per second, at least 30,000 droplets per second, at least40,000 droplets per second, at least 50,000 droplets per second, atleast 60,000 droplets per second, at least 70,000 droplets per second,at least 80,000 droplets per second, at least 90,000 droplets persecond, at least 100,000 droplets per second.
 29. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 1, wherein said species of mammal comprises a bovinemammal.
 30. A method of isolating X-chromosome bearing sperm cells andY-chromosome bearing sperm cells as described in claim 1, wherein saidspecies of mammal comprises an equine mammal.
 31. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 1, wherein said species of mammal comprises an ovinemammal.
 32. An apparatus to isolate X-chromosome bearing sperm cells andY-chromosome bearing sperm cells, comprising: a. sperm cells having asperm heads, wherein said sperm heads have a difference in volumebetween X-chromosome bearing sperms cells and Y-chromosome bearing spermcells; b. a beam of electromagnetic radiation having initial waveformcharacteristics differentially responsive to said difference in volumebetween X-chromosome bearing sperms cells and Y-chromosome bearing spermcells; c. a detector responsive to altered waveform characteristics ofsaid beam of electromagnetic radiation; d. an analyzer coupled to saiddetector, wherein said analyzer differentiates between said differencein volume between said X-chromosome bearing sperms cells and saidY-chromosome bearing sperm cells based upon said altered waveformcharacteristics.
 33. An apparatus to isolate X-chromosome bearing spermcells and Y-chromosome bearing sperm cells as described in claim 32,further comprising a fluid stream into which said sperm cells areintroduced.
 34. An apparatus to isolate X-chromosome bearing sperm cellsand Y-chromosome bearing sperm cells as described in claim 33, furthercomprising droplets breaking off from said fluid stream a pluralityhaving one of said sperm cells entrained.
 35. An apparatus to isolateX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 34, wherein said droplets breaking off from saidfluid stream have sufficient size to encapsulate said one of said spermcells.
 36. An apparatus to isolate X-chromosome bearing sperm cells andY-chromosome bearing sperm cells as described in claim 35, furthercomprising a nozzle having an orifice of about 100 micrometers indiameter.
 37. An apparatus to isolate X-chromosome bearing sperm cellsand Y-chromosome bearing sperm cells as described in claim 36, furthercomprising a droplet charger coupled to said analyzer, wherein saiddroplets receive a charge differentially based upon said difference involume between X-chromosome bearing sperms cells and Y-chromosomebearing sperm cells.
 38. An apparatus to isolate X-chromosome bearingsperm cells and Y-chromosome bearing sperm cells as described in claim37, further comprising a droplet separator, wherein said dropletseparator separates said droplet based upon charge of said droplet. 39.An apparatus to isolate X-chromosome bearing sperm cells andY-chromosome bearing sperm cells as described in claim 38, furthercomprising at least one collection container in which dropletscontaining said X-chromosome bearing sperm cells are collected as anX-chromosome bearing population.
 40. An apparatus to isolateX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 38, further comprising at least one collectioncontainer in which droplets containing Y-chromosome bearing sperm cellsare collected as a Y-chromosome bearing population.
 41. An apparatus toisolate X-chromosome bearing sperm cells and Y-chromosome bearing spermcells as described in claims 38 or 39, wherein said X-chromosome bearingpopulation and said Y-chromosome bearing population of said sperm cellshave a purity selected from the group consisting of between 90% to about100%, between about 91% to about 100%, between about 92% to about 100%,between about 93% to about 100%, between about 94% to about 100%,between about 95% to about 100%, between about 96% to about 100%,between about 97% to about 100%, between about 98% to about 100%,between about 99% to about 100%.
 42. An apparatus to isolateX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 41, further comprising a separable event rate whereinsaid separable event rate is selected from the group consisting of atleast 5000 separable events per second, at least 6000 separable eventsper second, at least 7000 separable events per second, at least 8000separable events per second, at least 9000 separable events per second,at least 10,000 separable events per second, at least 11,000 separableevents per second, at least 12,000 separable events per second, at least13,000 separable events per second, at least 14,000 separable events persecond, at least 15,000 separable events per second, at least 16,000separable events per second, at least 17,000 separable events persecond, at least 18,000 separable events per second, at least 19,000separable events per second, at least 20,000 separable events persecond, at least 21,000 separable events per second.
 43. An apparatus toisolate X-chromosome bearing sperm cells and Y-chromosome bearing spermcells as described in claim 42, further comprising a separation rateselected from the group consisting of at least 500 separations persecond, at least 1,000 separations per second, at least 2,000separations per second, at least 3,000 separations per second, at least4,000 separations per second, at least 5,000 separations per second, atleast 6,000 separations per second, at least 7,000 separations persecond, at least 8,000 separations per second, at least 9,000separations per second, at least 10,000 separations per second, 11,000separations per second.
 44. An apparatus to isolate X-chromosome bearingsperm cells and Y-chromosome bearing sperm cells as described in claim43, further comprising a droplet formation rate selected from the groupconsisting of at least 10,000 droplets per second, at least 20,000droplets per second, at least 30,000 droplets per second, at least40,000 droplets per second, at least 50,000 droplets per second, atleast 60,000 droplets per second, at least 70,000 droplets per second,at least 80,000 droplets per second, at least 90,000 droplets persecond, at least 100,000 droplets per second.
 45. An apparatus toisolate X-chromosome bearing sperm cells and Y-chromosome bearing spermcells as described in claim 44, wherein said detector comprises at leastone photomultiplier tube, and wherein said at least one photomultipliertube converts said electromagnetic radiation into at least one signal,and wherein said photomultiplier tube has a typical operation voltagerange, and wherein said photomultiplier tube is operated outside saidtypical operation voltage range.
 46. An apparatus to isolateX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 45, wherein said typical operation voltage range ofsaid photomultiplier tube is from about 400 volts to about 999 volts.47. An apparatus to isolate X-chromosome bearing sperm cells andY-chromosome bearing sperm cells as described in claim 46, wherein saidphotomultiplier tube is operated in a range from about 0 volts to about300 volts.
 48. An apparatus to isolate X-chromosome bearing sperm cellsand Y-chromosome bearing sperm cells as described in claim 41, whereinsaid species of mammal comprises a bovine mammal.
 49. An apparatus toisolate X-chromosome bearing sperm cells and Y-chromosome bearing spermcells as described in claim 41, wherein said species of mammal comprisesan equine mammal.
 50. An apparatus to isolate X-chromosome bearing spermcells and Y-chromosome bearing sperm cells as described in claim 41,wherein said species of mammal comprises an ovine mammal.
 51. A methodof isolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells, comprising the steps of: a. collecting sperm cells from amale of a species of mammal; a. assessing a volume of a capsulecontaining the nuclear DNA of said sperm cells; c. differentiatingbetween said X-chromosome bearing sperm cells and said Y-chromosomebearing sperm cells based upon said volume of said capsule containingsaid nuclear DNA of said sperm cells;
 52. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 51, wherein said step of assessing the volume of acapsule containing said DNA comprises determining a phase shift in saidelectromagnetic radiation.
 53. A method of isolating X-chromosomebearing sperm cells and Y-chromosome bearing sperm cells as described inclaim 52, wherein said step of assessing the volume of a capsulecontaining said DNA comprises: a. generating a beam electromagneticradiation having initial waveform characteristics; b. traversing saidvolume of said capsule containing said DNA with said beam ofelectromagnetic radiation having initial waveform characteristics; c.altering said initial waveform characteristics by traversing said volumeof said capsule; and d. analyzing altered wave form characteristics ofsaid beam of electromagnetic radiation.
 54. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 53, wherein said step of altering said initial waveform characteristics by traversing said volume of said capsule comprisesshifting phase of said initial waveform characteristics of said beam ofelectromagnetic radiation.
 55. A method of isolating X-chromosomebearing sperm cells and Y-chromosome bearing sperm cells as described inclaim 54, further comprising the step of superimposing said initialwaveform characteristics and phase shifted waveform characteristics. 56.A method of isolating X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells as described in claim 55, further comprising thestep of comparing an intensity of said initial waveform characteristicswith superimposed waveform characteristics.
 57. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 56, further comprising the step of determining saidvolume of said capsule containing said DNA based upon the difference insaid intensity between said superimposed waveform characteristics andsaid initial waveform characteristics.
 58. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 57, further comprising the steps of: a. introducingsaid sperm cells into a fluid stream; b. forming droplets a pluralityhaving one of said sperm cells entrained; c. charging each of saiddroplets differentially based upon the assessed volume of said capsulecontaining the nuclear DNA of said sperm cells; d. deflecting each ofsaid droplets; e. differentially collecting each of said droplets basedupon said volume of said capsule containing said DNA; and f. generatingX-chromosome bearing and Y-chromosome bearing populations of said spermcells.
 59. A method of isolating X-chromosome bearing sperm cells andY-chromosome bearing sperm cells as described in claim 58, furthercomprising the step of maintaining said amount of DNA within the nucleusof said sperm cells unstained.
 60. a method of isolating X-chromosomebearing sperm cells and Y-chromosome bearing sperm cells as described inclaim 59, further comprising the step of forming droplets havingsufficient size to encapsulate said sperm cells.
 61. a method ofisolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells as described in claim 60, further comprises the step ofejecting said fluid stream from a nozzle having an orifice, wherein saidorifice has a diameter of 100 micrometers.
 62. a method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 58, further comprises the steps of: a. detecting asignal having sperm cell orientation characteristics; b. comparing saidsignal having sperm cell orientation characteristics to a signal havingoriented sperm cell characteristics; c. determining said sperm cell hasunoriented sperm cell characteristics; and d. differentially collectingsaid sperm cell having unoriented sperm cell characteristics.
 63. Amethod of isolating X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells as described in claims 58 or 62, wherein purity ofsaid X-chromosome bearing and said Y-chromosome bearing populations ofsaid sperm cells is selected from the group consisting of between 90% toabout 100%, between about 91% to about 100%, between about 92% to about100%, between about 93% to about 100%, between about 94% to about 100%,between about 95% to about 100%, between about 96% to about 100%,between about 97% to about 100%, between about 98% to about 100%,between about 99% to about 100%.
 64. A method of isolating X-chromosomebearing sperm cells and Y-chromosome bearing sperm cells as described inclaim 63, further comprising the step of establishing a separable eventrate wherein said separable event rate is selected from the groupconsisting of at least 5000 separable events per second, at least 6000separable events per second, at least 7000 separable events per second,at least 8000 separable events per second, at least 9000 separableevents per second, at least 10,000 separable events per second, at least11,000 separable events per second, at least 12,000 separable events persecond, at least 13,000 separable events per second, at least 14,000separable events per second, at least 15,000 separable events persecond, at least 16, 000 separable events per second, at least 17,000separable events per second, at least 18,000 separable events persecond, at least 19,000 separable events per second, at least 20,000separable events per second, at least 21,000 separable events persecond.
 65. A method of isolating X-chromosome bearing sperm cells andY-chromosome bearing sperm cells as described in claim 64, wherein saidstep of separating said sperm cells comprises a separation rate selectedfrom the group consisting of at least 500 separations per second, atleast 1,000 separations per second, at least 2,000 separations persecond, at least 3,000 separations per second, at least 4,000separations per second, at least 5,000 separations per second, at least6,000 separations per second, at least 7,000 separations per second, atleast 8,000 separations per second, at least 9,000 separations persecond, at least 10,000 separations per second.
 66. A method ofisolating X-chromosome bearing sperm cells and Y-chromosome bearingsperm cells as described in claim 65, wherein said step of formingdroplets each having one of said sperm cells entrained comprises adroplet formation rate selected from the group consisting of at least10,000 droplets per second, at least 20,000 droplets per second, atleast 30,000 droplets per second, at least 40,000 droplets per second,at least 50,000 droplets per second, at least 60,000 droplets persecond, at least 70,000 droplets per second, at least 80,000 dropletsper second, at least 90,000 droplets per second, at least 100,000droplets per second.
 67. A method of isolating X-chromosome bearingsperm cells and Y-chromosome bearing sperm cells as described in claim51, wherein said species of mammal comprises a bovine mammal.
 68. Amethod of isolating X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells as described in claim 51, wherein said species ofmammal comprises an equine mammal.
 69. A method of isolatingX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells asdescribed in claim 51, wherein said species of mammal comprises an ovinemammal.
 70. A method differentiating particles, comprising the steps of:a. collecting asymmetric particles; b. establishing said fluid streamwith a flow cytometer; c. introducing said asymmetric particles intosaid fluid stream; d. orienting said asymmetric particles with respectto a detector; e. detecting light having characteristics differentiallyresponsive to asymmetric particle orientation to said detector; g.converting said light having characteristics differentially responsiveto asymmetric particle orientation into at least one signal containingasymmetric particle orientation information; h. analyzing asymmetricparticle orientation information; and i. determining orientation of saidasymmetric particles with respect to said detector.
 71. A methoddifferentiating particles as described in claim 70, wherein said fluidstream comprises a sheath fluid
 72. A method differentiating particlesas described in claim 70, wherein said detector comprises aphotomultiplier tube.
 73. A method differentiating particles asdescribed in claim 72, wherein said light comprises florescent light.74. A method differentiating particles as described in claim 73, whereinsaid florescent light emits from a light emission material bound to saidasymmetric particles.
 75. A method differentiating particles asdescribed in claim 70, wherein said step of analyzing asymmetricparticle orientation information comprises the steps of: a. plottingelectronic signal over a period of time; b. integrating the areas ofplots corresponding to said electronic signal; and c. comparingintegrations of said plots to integrations of oriented asymmetricparticles.
 76. A method differentiating particles as described in claim70, further comprising differentially collecting said asymmetricparticles based upon determined orientation of said asymmetric particleswith respect to said detector.
 77. A method differentiating particles asdescribed in claim 70, wherein said asymmetric particles are sperm cellsfrom a male of a species of mammal.
 78. A method differentiatingparticles as described in claim 70, further comprising the steps of: a.assessing the volume of a capsule containing the nuclear DNA of saidsperm cells; and b. differentiating between said X-chromosome bearingsperm cells and said Y-chromosome bearing sperm cells based upon thevolume of said capsule containing the nuclear DNA of said sperm cell.79. A method differentiating particles as described in claim 78, whereinsaid capsule containing said DNA comprises a sperm head.
 80. A methoddifferentiating particles as described in claim 78, wherein said step ofassessing the volume of a capsule containing said DNA comprises: a.generating a beam of electromagnetic radiation having initial waveformcharacteristics; b. traversing said volume of said capsule containingsaid DNA with said beam of electromagnetic radiation having initialwaveform characteristics; c. altering said initial wave formcharacteristics of said beam of electromagnetic radiation by traversingsaid volume of said capsule; and d. analyzing altered wave formcharacteristics.
 81. A method differentiating particles as described inclaim 80, wherein said step of altering said wave form characteristicsby traversing said volume of said capsule comprises shifting phase ofsaid initial waveform characteristics.
 82. A method differentiatingparticles as described in claim 81, further comprising the step ofsuperimposing said initial waveform characteristics and said phaseshifted waveform characteristics.
 83. A method differentiating particlesas described in claim 82, further comprising the step of comparing aintensity of said initial waveform characteristics and superimposedwaveform characteristics.
 84. A method differentiating particles asdescribed in claim 83, further comprising the step of determining saidvolume of said capsule containing said DNA based upon the difference insaid intensity.
 85. A method differentiating particles as described inclaim 80, further comprising the steps of: a. forming droplets aplurality having one of said sperm cells entrained; b. charging each ofsaid droplets differentially based upon the determined volume of saidcapsule containing the nuclear DNA of said sperm cells; c. deflectingeach of said droplets; and d. collecting each of said droplets basedupon charge of said droplet; and e. generating X-chromosome bearing andY-chromosome bearing populations of said sperm cells.
 86. A methoddifferentiating particles as described in claim 77, further comprisingthe steps of: a. forming droplets a plurality having one of said spermcells entrained; b. charging each of said droplets differentially basedupon an amount of nuclear DNA of said sperm cells; c. deflecting each ofsaid droplets; d. collecting each of said droplets based upon charge ofsaid droplet; and e. generating X-chromosome bearing and Y-chromosomebearing populations of said sperm cells.
 87. A method differentiatingparticles as described in claims 85 or 86, wherein said step of formingdroplets a plurality having one of said sperm cells entrained comprisesforming droplets having sufficient size to encapsulate said sperm cell,wherein said sperm cell comprises an intact live sperm cell having atail.
 88. A method differentiating particles as described in claim 87,further comprises the step of ejecting said fluid stream from a nozzlehaving an orifice, wherein said orifice has a diameter of 100micrometers.
 89. A method differentiating particles as described inclaim 88, wherein said X-chromosome bearing and said Y-chromosomebearing populations of sperm cells have a purity selected from the groupconsisting of between 90% to about 100%, between about 91% to about100%, between about 92% to about 100%, between about 93% to about 100%,between about 94% to about 100%, between about 95% to about 100%,between about 96% to about 100%, between about 97% to about 100%,between about 98% to about 100%, between about 99% to about 100%.
 90. Amethod differentiating particles as described in claim 88, furthercomprising the step of establishing a separable event rate wherein saidseparable event rate is selected from the group consisting of at least5000 separable events per second, at least 6000 separable events persecond, at least 7000 separable events per second, at least 8000separable events per second, at least 9000 separable events per second,at least 10,000 separable events per second, at least 11,000 separableevents per second, at least 12,000 separable events per second, at least13,000 separable events per second, at least 14,000 separable events persecond, at least 15,000 separable events per second, at least 16, 000separable events per second, at least 17,000 separable events persecond, at least 18,000 separable events per second, at least 19,000separable events per second, at least 20,000 separable events persecond, at least 21,000 separable events per second.
 91. A methoddifferentiating particles as described in claim 88, wherein said step ofseparating said sperm cells comprises a separation rate selected fromthe group consisting of at least 500 separations per second, at least1,000 separations per second, at least 2,000 separations per second, atleast 3,000 separations per second, at least 4,000 separations persecond, at least 5,000 separations per second, at least 6,000separations per second, at least 7,000 separations per second, at least8,000 separations per second, at least 9,000 separations per second, atleast 10,000 separations per second.
 92. A method differentiatingparticles as described in claim 88, wherein said step of formingdroplets each having one of said sperm cells entrained comprises adroplet formation rate selected from the group consisting of at least10,000 droplets per second, at least 20,000 droplets per second, atleast 30,000 droplets per second, at least 40,000 droplets per second,at least 50,000 droplets per second, at least 60,000 droplets persecond, at least 70,000 droplets per second, at least 80,000 dropletsper second, at least 90,000 droplets per second, at least 100,000droplets per second.
 93. A method differentiating particles as describedin claim 77, wherein said species of mammal comprises a bovine mammal.94. A method differentiating particles as described in claim 77, whereinsaid species of mammal comprises an equine mammal.
 95. A methoddifferentiating particles as described in claim 77, wherein said speciesof mammal comprises an ovine mammal.
 96. A particle differentiationapparatus, comprising: a. at least one asymmetric particle havingorientation characteristics within a fluid stream; b. a irradiationsource generating an irradiation beam responsive to said asymmetricparticle; c. optics to focus said irradiation beam; d. a light emissionmaterial coupled to said asymmetric particle, wherein said lightemission material emits light in response to said irradiation beam; e. adetector deferentially responsive to said light emitted from said lightemission material based upon said particle orientation characteristics;and f. an analyzer coupled to said detector, wherein said analyzerdifferentiates said asymmetric particles based upon orientation withinsaid fluid stream.
 97. A particle differentiation apparatus as describedin claim 96, wherein said at least one asymmetrical particle comprise atleast one sperm cell.
 98. A particle differentiation apparatus asdescribed in claim 96, wherein said optics focus a beam pattern having aheight of about equal to the length of said asymmetrical particle alongthe longitudinal axis to about three times the length of saidasymmetrical particle along the longitudinal axis;
 99. A particledifferentiation apparatus as described in claim 98, wherein said atleast one sperm cell has a head having a length along the longitudinalaxis of about nine micrometers, and wherein said irradiation pattern hasa height of about 20 micrometers.
 100. A particle differentiationapparatus as described in claim 96, wherein said at least one asymmetricparticle having orientation characteristics further comprises at leastone particle differentiation characteristic.
 101. A particledifferentiation apparatus as described in claim 96, wherein said lightemission material bound to said at least one asymmetric particlecomprises a stain bound to nuclear DNA of sperm cells of a male of aspecies of mammal.
 102. A particle differentiation apparatus asdescribed in claim 101, wherein said particle differentiationcharacteristic comprises a difference in amount of said stain bound tosaid nuclear DNA of X-chromosome bearing sperm cells and said nuclearDNA of Y-chromosome bearing sperm cells.
 103. A particle differentiationapparatus as described in claim 96, wherein said detector comprises atleast one photomultiplier tube, and wherein said photomultiplier tubehas a typical operation voltage range, and wherein said photomultipliertube is operated outside said typical operation voltage range.
 104. Aparticle differentiation apparatus as described in claim 103, whereinsaid typical operation voltage range of said photomultiplier tube isfrom about 400 volts to about 999 volts.
 105. A particle differentiationapparatus as described in claim 104, wherein said photomultiplier tubeis operated in a range from about 0 volts to about 300 volts.
 106. Aparticle differentiation apparatus as described in claim 96, furthercomprising droplets breaking off from said fluid stream a pluralityhaving at least one of said asymmetric particles entrained.
 107. Aparticle differentiation apparatus as described in claim 106, whereinsaid droplets breaking off from said fluid stream have sufficient sizeto encapsulate one of said sperm cells, wherein said sperm cellscomprise intact live sperm cells having a tail.
 108. A particledifferentiation apparatus as described in claim 107, further comprisinga nozzle having an orifice of about 100 micrometers in diameter.
 109. Aparticle differentiation apparatus as described in claim 106, furthercomprising a droplet charger coupled to said analyzer, wherein saiddroplets receive a charge differentially based upon said difference inamount of said stain bound the nuclear DNA of X-chromosome sperm cellsand the nuclear DNA of Y-chromosome bearing sperm cells.
 110. A particledifferentiation apparatus as described in claim 109, further comprisinga droplet charger coupled to said analyzer, wherein said dropletsreceive a charge differentially based upon said difference in volume ofsaid particles, and wherein said difference in volume of said particlescomprises a difference between X-chromosome bearing sperm cells andY-chromsome bearing sperm cells
 111. A particle differentiationapparatus as described in claim 110, further comprising a dropletseparator, wherein said droplet separator separates said droplet basedupon charge of said droplet.
 112. A particle differentiation apparatusas described in claim 111, further comprising at least one collectioncontainer in which droplets containing said X-chromosome bearing spermcells are collected as an X-chromosome bearing population.
 113. Aparticle differentiation apparatus as described in claim 111, furthercomprising at least one collection container in which dropletscontaining Y-chromosome bearing sperm cells are collected as aY-chromosome bearing population.
 114. A particle differentiationapparatus as described in claim 112 or 113, wherein said X-chromosomebearing population and said Y-chromosome bearing population of saidsperm cells are selected from the group consisting of between 90% toabout 100%, between about 91% to about 100%, between about 92% to about100%, between about 93% to about 100%, between about 94% to about 100%,between about 95% to about 100%, between about 96% to about 100%,between about 97% to about 100%, between about 98% to about 100%,between about 99% to about 100%.
 115. A particle differentiationapparatus as described in claim 114, further comprising the step ofestablishing a separable event rate wherein said separable event rate isselected from the group consisting of at least 5000 separable events persecond, at least 6000 separable events per second, at least 7000separable events per second, at least 8000 separable events per second,at least 9000 separable events per second, at least 10,000 separableevents per second, at least 11,000 separable events per second, at least12,000 separable events per second, at least 13,000 separable events persecond, at least 14,000 separable events per second, at least 15,000separable events per second, at least 16, 000 separable events persecond, at least 17,000 separable events per second, at least 18,000separable events per second, at least 19,000 separable events persecond, at least 20,000 separable events per second, at least 21,000separable events per second.
 116. A particle differentiation apparatusas described in claim 115, wherein said step of separating said spermcells comprises a separation rate selected from the group consisting ofat least 500 separations per second, at least 1,000 separations persecond, at least 2,000 separations per second, at least 3,000separations per second, at least 4,000 separations per second, at least5,000 separations per second, at least 6,000 separations per second, atleast 7,000 separations per second, at least 8,000 separations persecond, at least 9,000 separations per second, at least 10,000separations per second, 11,000 separations per second.
 117. A particledifferentiation apparatus as described in claim 116, wherein said stepof forming droplets each having one of said sperm cells entrainedcomprises a droplet formation rate selected from the group consisting ofat least 10,000 droplets per second, at least 20,000 droplets persecond, at least 30,000 droplets per second, at least 40,000 dropletsper second, at least 50,000 droplets per second, at least 60,000droplets per second, at least 70,000 droplets per second, at least80,000 droplets per second, at least 90,000 droplets per second, atleast 100,000 droplets per second.
 118. A particle differentiationapparatus as described in claim 102, wherein said species of mammalcomprises a bovine mammal.
 119. A particle differentiation apparatus asdescribed in claim 102, wherein said species of mammal comprises anequine mammal.
 120. A particle differentiation apparatus as described inclaim 102, wherein said species of mammal comprises an ovine mammal.121. A particle differentiation apparatus, comprising: a. a fluidstream; b. intact live sperm cells introduced into said fluid stream; c.a nozzle having a orifice through which said fluid stream exits; d. anoscillator responsive to said fluid stream; and e. droplets breaking offfrom said fluid stream, wherein a plurality of said droplets entrainsaid intact live sperm cells, and wherein said droplets have sufficientsize to encapsulate one of said live sperm cells.
 122. A particledifferentiation apparatus as described in claim 121, wherein saidorifice has a diameter of 100 micrometers.
 123. A particledifferentiation apparatus as described in claim 122, wherein saiddroplets breaking off from said fluid stream break off at a rateselected from the group consisting of at least 10,000 droplets persecond, at least 20,000 droplets per second, at least 30,000 dropletsper second, at least 40,000 droplets per second, at least 50,000droplets per second, at least 60,000 droplets per second, at least70,000 droplets per second, at least 80,000 droplets per second, atleast 90,000 droplets per second, at least 100,000 droplets per second.124. A particle differentiation apparatus as described in claim 123,further comprising a light emission source generating light.
 125. Aparticle differentiation apparatus as described in claim 124, furthercomprising a detector responsive to said light.
 126. A particledifferentiation apparatus as described in claim 125, wherein said intactlive sperm cells have at least one sex differentiation characteristic.127. A particle differentiation apparatus as described in claim 126,wherein said at least one sex differentiation characteristic comprises avolume difference of sperm cell heads of said intact live sperm cells.128. A particle differentiation apparatus as described in claim 127,wherein said light emission source emits a beam of electromagneticradiation, and wherein said beam of electromagnetic radiation traversessaid volume of said sperm cell heads, and wherein said beam ofelectromagnetic radiation has initial waveform characteristicsdifferentially responsive to said difference in volume of said spermcell heads.
 129. A particle differentiation apparatus as described inclaim 128, further comprising an analyzer responsive to said detector,wherein said analyzer differentiates between said intact live spermcells based upon said volume difference of said sperm cell heads.
 130. Aparticle differentiation apparatus as described in claim 129, whereinsaid volume difference of said sperm cell heads in volume comprises adifference between X-chromosome bearing live intact sperm cells andY-chromsome bearing live intact sperm cells.
 131. A particledifferentiation apparatus as described in claim 126, an irradiationsource generating an irradiation beam responsive to said intact livesperm cells.
 132. A particle differentiation apparatus as described inclaim 131, further comprising optics, wherein said optics focus anirradiation beam pattern having a height equal to about the length ofsaid sperm heads along the longitudinal axis to about three times thelength of said sperm head of saids along the longitudinal axis.
 133. Aparticle differentiation apparatus as described in claim 132, whereinsaid sperm heads have a length along the longitudinal axis of about ninemicrometers, and wherein said irradiation pattern has a height of about20 micrometers.
 134. A particle differentiation apparatus as describedin claim 131, wherein said at least one sex differentiationcharacteristic comprises a difference in amount of nuclear DNA of saidintact live sperm cells, and wherein said light emission sourcecomprises a light emission material bound to nuclear DNA, and whereinsaid light emission material emits light differentially based upon saiddifference in amount of said nuclear DNA, and wherein and wherein saiddetector generates at least one signal in response to said light.
 135. Aparticle differentiation apparatus as described in claim 134, furthercomprising an analyzer responsive to said detector, wherein saidanalyzer differentiates between said at least one intact live sperm cellbased upon said difference in amount of nuclear DNA.
 136. A particledifferentiation apparatus as described in claim 135, wherein saiddifference in said amount of said nuclear DNA comprises a differencebetween X-chromosome bearing sperm cells and Y-chromosome bearing spermcells.
 137. A particle differentiation apparatus as described in claims136, further comprising a droplet charger coupled to said analyzer,wherein said droplet charger generates a charge on said dropletsdifferentially based upon said difference in amount of said nuclear DNA.138. A particle differentiation apparatus as described in claim 129,further comprising a droplet charger coupled to said analyzer, whereinsaid droplet charger generates a charge on said droplets differentiallybased upon said volume difference of said sperm cell heads, and whereinsaid volume difference of said sperm cell heads comprises a differencebetween X-chromosome bearing sperm cells and Y-chromsome bearing spermcells.
 139. A particle differentiation apparatus as described in claims137 or 138, further comprising a droplet separator, wherein said dropletseparator separates said droplets based upon charge of said droplets.140. A particle differentiation apparatus as described in claim 139,further comprising at least one collection container in which dropletscontaining said X-chromosome bearing sperm cells are collected as anX-chromosome bearing population.
 141. A particle differentiationapparatus as described in claim 139, further comprising at least onecollection container in which droplets containing Y-chromosome bearingsperm cells are collected as a Y-chromosome bearing population.
 142. Aparticle differentiation apparatus as described in claim 125, whereinsaid detector comprises at least one photomultiplier tube, and whereinsaid at least one photomultiplier tube converts light from said lightemission source into at least one signal, and wherein saidphotomultiplier tube has a typical operation voltage range, and whereinsaid photomultiplier tube is operated outside said typical operationvoltage range.
 143. A particle differentiation apparatus as described inclaim 142, wherein said typical operation voltage range of saidphotomultiplier tube is from about 400 volts to about 999 volts.
 144. Aparticle differentiation apparatus as described in claim 143, whereinsaid photomultiplier tube is operated in a range from about 0 volts toabout 300 volts. 145 A particle differentiation apparatus as describedin claims 140 or 141, wherein said X-chromosome bearing population andsaid Y-chromosome bearing population of said sperm cells have a purityselected from the group consisting of between 90% to about 100%, betweenabout 91% to about 100%, between about 92% to about 100%, between about93% to about 100%, between about 94% to about 100%, between about 95% toabout 100%, between about 96% to about 100%, between about 97% to about100%, between about 98% to about 100%, between about 99% to about 100%.146. A particle differentiation apparatus as described in claim 145,further comprising the step of establishing a separable event ratewherein said separable event rate is selected from the group consistingof at least 5000 separable events per second, at least 6000 separableevents per second, at least 7000 separable events per second, at least8000 separable events per second, at least 9000 separable events persecond, at least 10,000 separable events per second, at least 11,000separable events per second, at least 12,000 separable events persecond, at least 13,000 separable events per second, at least 14,000separable events per second, at least 15,000 separable events persecond, at least 16, 000 separable events per second, at least 17,000separable events per second, at least 18,000 separable events persecond, at least 19,000 separable events per second, at least 20,000separable events per second, at least 21,000 separable events persecond.
 147. A particle differentiation apparatus as described in claim146, wherein said step of separating said sperm cells comprises aseparation rate selected from the group consisting of at least 500separations per second, at least 1,000 separations per second, at least2,000 separations per second, at least 3,000 separations per second, atleast 4,000 separations per second, at least 5,000 separations persecond, at least 6,000 separations per second, at least 7,000separations per second, at least 8,000 separations per second, at least9,000 separations per second, at least 10,000 separations per second,11,000 separations per second.
 148. A particle differentiationapparatus, comprising: a. particles having at least one particledifferentiation characteristic; b. a light emission sourcedifferentially responsive to said particle differentiationcharacteristics; c. at least one photomultiplier tube, wherein said atleast one photomultiplier tube converts light from said light emissionsource into at least one signal, and wherein said photomultiplier tubehas a typical operation voltage range, and wherein said photomultipliertube is operated outside said typical operation voltage range; and d. ananalyzer responsive to said at least one signal which differentiatesparticles based upon said particle differentiation characteristics. 149.A particle differentiation apparatus as described in claim 148, whereinsaid typical operation voltage range of said photomultiplier tube isfrom about 400 volts to about 999 volts.
 150. A particle differentiationapparatus as described in claim 149, wherein said photomultiplier tubeis operated in a range from about 0 volts to about 300 volts.
 151. Aparticle differentiation apparatus as described in claim 148, furthercomprising a irradiation source generating an irradiation beamresponsive to said particles.
 152. A particle differentiation apparatusas described in claim 151, wherein said light emission source comprisesa light emission material bound to said particles which emits said lightin response to said irradiation beam.
 153. A particle differentiationapparatus as described in claim 152, wherein said light emissionmaterial bound to said particles comprises a stain bound to the nuclearDNA of sperm cells.
 154. A particle differentiation apparatus asdescribed in claim 153, wherein said particle differentiationcharacteristic comprises a difference in amount of said stain bound thenuclear DNA of X-chromosome sperm cells and the nuclear DNA ofY-chromosome bearing sperm cells.
 155. A particle differentiationapparatus as described in claim 152, wherein said particle comprises anasymmetric particle.
 156. A particle differentiation apparatus asdescribed in claim 155, further comprising optics to focus saidirradiation beam responsive to said particle, wherein said optics focusa irradiation pattern having a height of about equal to the length ofsaid asymmetrical particle along the longitudinal axis to about threetimes the length of said asymmetrical particle along the longitudinalaxis.
 157. A particle differentiation apparatus as described in claim156, further comprising a fluid stream into which said particles areintroduced.
 158. A particle differentiation apparatus as described inclaim 157, wherein said at least one particle differentiationcharacteristic comprises orientation of said asymmetric particle withinsaid fluid stream, and wherein said photomultiplier tube isdeferentially responsive to said light emitted from said light emissionmaterial based upon said particle orientation characteristics, andwherein said analyzer coupled to said photomultiplier tubedifferentiates between said asymmetric particles based upon orientationwithin said fluid stream.
 159. A particle differentiation apparatus asdescribed in claim 158, wherein said asymmetrical particles comprisesperm cells having sperm cell heads.
 160. A particle differentiationapparatus as described in claim 159, wherein said sperm cell heads ofsaid sperm cell have a length along the longitudinal axis of about ninemicrometers, and wherein said irradiation pattern has a height of about20 micrometers.
 161. A particle differentiation apparatus as describedin claim 148, wherein said particles have a volume, and wherein said atleast one particle differentiation characteristic comprises a volumedifference of said particles.
 162. A particle differentiation apparatusas described in claim 161, wherein said light emission source emits abeam of electromagnetic radiation, and wherein said beam ofelectromagnetic radiation traverses said volume of said particle, andwherein said beam of electromagnetic radiation has initial waveformcharacteristics altered by said volume of said particles.
 163. Aparticle differentiation apparatus as described in claim 162, whereinsaid photomultiplier tube is responsive to said waveform characteristicsaltered by said volume of said particles.
 164. A particledifferentiation apparatus as described in claim 163, wherein saidanalyzer responsive to said at least one signal which differentiatesparticles based upon said particle differentiation characteristicsdifferentiates between said particles based upon said difference involume.
 165. A particle differentiation apparatus as described in claim164, wherein said volume difference comprises a difference betweenX-chromosome bearing and Y-chromsome bearing sperm cells.
 166. Aparticle differentiation apparatus as described in claim 160, furthercomprising droplets breaking off from said fluid stream a pluralityhaving one of said sperm cells entrained.
 167. A particledifferentiation apparatus as described in claim 165, further comprisingdroplets breaking off from said fluid stream a plurality having one ofsaid sperm cells entrained.
 168. A particle differentiation apparatus asdescribed in claim 166, wherein said droplets breaking off from saidfluid stream have sufficient size to encapsulate said one of said spermcells.
 169. A particle differentiation apparatus as described in claim168, further comprising a nozzle having an orifice of about 100micrometers in diameter.
 170. A particle differentiation apparatus asdescribed in claim 166, further comprising a droplet charger coupled tosaid analyzer, wherein said droplets receive a charge differentiallybased upon said difference in amount of said stain bound the nuclear DNAof X-chromosome sperm cells and the nuclear DNA of Y-chromosome bearingsperm cells.
 171. A particle differentiation apparatus as described inclaim 167, further comprising a droplet charger coupled to saidanalyzer, wherein said droplets receive a charge differentially basedupon said volume difference of said sperm cell heads, and wherein saiddifference in volume of said sperm cell heads comprise a differencebetween X-chromosome bearing sperm cells and Y-chromsome bearing spermcells
 172. A particle differentiation apparatus as described in claim170, further comprising a droplet separator, wherein said dropletseparator separates said droplet based upon charge of said droplet. 173.A particle differentiation apparatus as described in claim 171, furthercomprising a droplet separator, wherein said droplet separator separatessaid droplet based upon charge of said droplet.
 174. A particledifferentiation apparatus as described in claim 172, further comprisingcollection containers responsive to said droplet separator, whereinX-chromosome bearing sperm cell and Y-chromosome bearing sperm cellpopulations are collected.
 175. A particle differentiation apparatus asdescribed in claim 173, further comprising collection containersresponsive to said droplet separator, wherein X-chromosome bearing spermcell and Y-chromosome bearing sperm cell populations are collected. 176.An apparatus for differentiating particles as described in claims 174 or175, wherein said X-chromosome bearing sperm cell population and saidY-chromosome bearing population of said sperm cells are selected fromthe group consisting of between 90% to about 100%, between about 91% toabout 100%, between about 92% to about 100%, between about 93% to about100%, between about 94% to about 100%, between about 95% to about 100%,between about 96% to about 100%, between about 97% to about 100%,between about 98% to about 100%, between about 99% to about 100%.
 177. Aparticle differentiation apparatus as described in claim 176, furthercomprising the step of establishing a separable event rate wherein saidseparable event rate is selected from the group consisting of at least5000 separable events per second, at least 6000 separable events persecond, at least 7000 separable events per second, at least 8000separable events per second, at least 9000 separable events per second,at least 10,000 separable events per second, at least 11,000 separableevents per second, at least 12,000 separable events per second, at least13,000 separable events per second, at least 14,000 separable events persecond, at least 15,000 separable events per second, at least 16, 000separable events per second, at least 17,000 separable events persecond, at least 18,000 separable events per second, at least 19,000separable events per second, at least 20,000 separable events persecond, at least 21,000 separable events per second.
 178. A particledifferentiation apparatus as described in claim 177, wherein said stepof separating said sperm cells comprises a separation rate selected fromthe group consisting of at least 500 separations per second, at least1,000 separations per second, at least 2,000 separations per second, atleast 3,000 separations per second, at least 4,000 separations persecond, at least 5,000 separations per second, at least 6,000separations per second, at least 7,000 separations per second, at least8,000 separations per second, at least 9,000 separations per second, atleast 10,000 separations per second, 11,000 separations per second. 179.A particle differentiation apparatus as described in claim 178, whereinsaid step of forming droplets each having one of said sperm cellsentrained comprises a droplet formation rate selected from the groupconsisting of at least 10,000 droplets per second, at least 20,000droplets per second, at least 30,000 droplets per second, at least40,000 droplets per second, at least 50,000 droplets per second, atleast 60,000 droplets per second, at least 70,000 droplets per second,at least 80,000 droplets per second, at least 90,000 droplets persecond, at least 100,000 droplets per second.
 180. A particledifferentiation apparatus as described in claim 179, wherein said spermcells comprise sperm cells from a bovine mammal.
 181. A particledifferentiation apparatus as described in claim 179, wherein said spermcells comprise sperm cells from an equine mammal.
 182. A particledifferentiation apparatus as described in claim 179, wherein said spermcells comprise sperm cells from an ovine mammal.
 183. A particledifferentiation apparatus, comprising: a. asymmetric particles; b. anirradiation source generating an irradiation beam responsive to saidasymmetric particles; c. optics to focus said irradiation beamresponsive to said asymmetrical particles, wherein said optics focus abeam pattern having a height of about equal to the length of saidasymmetrical particles along the longitudinal axis to about three timesthe length of said asymmetrical particles along the longitudinal axis;d. a light emission material coupled to said asymmetric particles,wherein said light emission material emits light in response to saidirradiation beam; e. a detector responsive to said light.
 184. Aparticle differentiation apparatus as described in claim 183, whereinsaid at least one asymmetrical particle comprise at least one spermcell.
 185. A particle differentiation apparatus as described in claim184, wherein said at least one sperm cell has a head having a lengthalong the longitudinal axis of about nine micrometers, and wherein saidirradiation pattern has a height of about 20 micrometers.
 186. Aparticle differentiation apparatus as described in claim 183, whereinsaid at least one asymmetric particle has at least one particledifferentiation characteristic.
 187. A particle differentiationapparatus as described in claim 186, further comprising a fluid stream.188. A particle differentiation apparatus as described in claim 187,wherein said at least one particle differentiation characteristiccomprises orientation of said asymmetric particle within said fluidstream, and wherein said detector is deferentially responsive to saidlight emitted from said light emission material based upon said particleorientation characteristics.
 189. A particle differentiation apparatusas described in claim 188, further comprising an analyzer coupled tosaid detector.
 190. A particle differentiation apparatus as described inclaim 189, wherein said analyzer differentiates between said asymmetricparticles based upon said orientation of said asymmetric particle withinsaid fluid stream.
 191. A particle differentiation apparatus asdescribed in claim 190, wherein said light emission material bound tosaid at least one asymmetric particle comprises a stain bound to thenuclear DNA of sperm cells.
 192. A particle differentiation apparatus asdescribed in claim 191, wherein said particle differentiationcharacteristic comprises a difference in amount of said stain bound thenuclear DNA of X-chromosome sperm cells and the nuclear DNA ofY-chromosome bearing sperm cells.
 193. A particle differentiationapparatus as described in claim 192, wherein said detector comprises atleast one photomultiplier tube, and wherein said photomultiplier tubehas a typical operation voltage range, and wherein said photomultipliertube is operated outside said typical operation voltage range.
 194. Aparticle differentiation apparatus as described in claim 193, whereinsaid typical operation voltage range of said photomultiplier tube isfrom about 400 volts to about 999 volts.
 195. A particle differentiationapparatus as described in claim 194, wherein said photomultiplier tubeis operated in a range from about 0 volts to about 300 volts.
 196. Aparticle differentiation apparatus as described in claim 193, furthercomprising droplets breaking off from said fluid stream each having oneof said particles entrained.
 197. A particle differentiation apparatusas described in claim 196, wherein said droplets breaking off from saidfluid stream have sufficient size to encapsulate said one of said spermcells, wherein said sperm cells comprise intact live sperm cells havingat least a head and a neck and a tail.
 198. A particle differentiationapparatus as described in claim 197, further comprising a nozzle havingan orifice of about 100 micrometers in diameter.
 199. A particledifferentiation apparatus as described in claim 196, further comprisinga droplet charger coupled to said analyzer, wherein said dropletsreceive a charge differentially based upon said difference in amount ofsaid stain bound the nuclear DNA of X-chromosome sperm cells and thenuclear DNA of Y-chromosome bearing sperm cells.
 200. A particledifferentiation apparatus as described in claim 199, further comprisinga droplet separator, wherein said droplet separator separates saiddroplet based upon charge of said droplet.
 201. A particledifferentiation apparatus as described in claim 200, further comprisingat least one collection container in which droplets containing saidX-chromosome bearing sperm cells are collected as an X-chromosomebearing population.
 202. A particle differentiation apparatus asdescribed in claim 200, further comprising at least one collectioncontainer in which droplets containing Y-chromosome bearing sperm cellsare collected as a Y-chromosome bearing population.
 203. A particledifferentiation apparatus as described in claims 201 or 202, whereinsaid X-chromosome bearing population and said Y-chromosome bearingpopulation of said sperm cells are selected from the group consisting ofbetween 90% to about 100%, between about 91% to about 100%, betweenabout 92% to about 100%, between about 93% to about 100%, between about94% to about 100%, between about 95% to about 100%, between about 96% toabout 100%, between about 97% to about 100%, between about 98% to about100%, between about 99% to about 100%.
 204. A particle differentiationapparatus as described in claim 203, further comprising the step ofestablishing a separable event rate wherein said separable event rate isselected from the group consisting of at least 5000 separable events persecond, at least 6000 separable events per second, at least 7000separable events per second, at least 8000 separable events per second,at least 9000 separable events per second, at least 10,000 separableevents per second, at least 11,000 separable events per second, at least12,000 separable events per second, at least 13,000 separable events persecond, at least 14,000 separable events per second, at least 15,000separable events per second, at least 16, 000 separable events persecond, at least 17,000 separable events per second, at least 18,000separable events per second, at least 19,000 separable events persecond, at least 20,000 separable events per second, at least 21,000separable events per second.
 205. An apparatus for differentiatingparticles as described in claim 204, wherein said step of separatingsaid sperm cells comprises a separation rate selected from the groupconsisting of at least 500 separations per second, at least 1,000separations per second, at least 2,000 separations per second, at least3,000 separations per second, at least 4,000 separations per second, atleast 5,000 separations per second, at least 6,000 separations persecond, at least 7,000 separations per second, at least 8,000separations per second, at least 9,000 separations per second, at least10,000 separations per second, 11,000 separations per second.
 206. Anapparatus for differentiating particles as described in claim 205,wherein said step of forming droplets each having one of said spermcells entrained comprises a droplet formation rate selected from thegroup consisting of at least 10,000 droplets per second, at least 20,000droplets per second, at least 30,000 droplets per second, at least40,000 droplets per second, at least 50,000 droplets per second, atleast 60,000 droplets per second, at least 70,000 droplets per second,at least 80,000 droplets per second, at least 90,000 droplets persecond, at least 100,000 droplets per second.
 207. A particledifferentiation apparatus as described in claim 206, wherein said spermcells comprise a bovine sperm cells.
 208. A particle differentiationapparatus as described in claim 206, wherein said sperm cells comprisesperm cells from an equine mammal.
 209. A particle differentiationapparatus as described in claim 206, wherein said sperm cells comprisesperm cells from an ovine mammal.